Psod Expression Units

ABSTRACT

The present invention relates to the use of nucleic acid sequences for regulating the transcription and expression of genes, the novel promoters and expression units themselves, methods for altering or causing the transcription rate and/or expression rate of genes, expression cassettes comprising the expression units, genetically modified microorganisms with altered or caused transcription rate and/or expression rate, and methods for preparing biosynthetic products by cultivating the genetically modified microorganisms.

The present invention relates to the use of nucleic acid sequences forregulating the transcription and expression of genes, the novelpromoters and expression units themselves, methods for altering orcausing the transcription rate and/or expression rate of genes,expression cassettes comprising the expression units, geneticallymodified microorganisms with altered or caused transcription rate and/orexpression rate, and methods for preparing biosynthetic products bycultivating the genetically modified microorganisms.

Various biosynthetic products such as, for example, fine chemicals, suchas, inter alia, amino acids, vitamins, but also proteins, are producedin cells by natural metabolic processes and are used in many branches ofindustry, including the cosmetics, feed, food and pharmaceuticalindustries. These substances, which are referred to collectively as finechemicals/proteins, comprise inter alia organic acids, bothproteinogenic and non-proteinogenic amino acids, nucleotides andnucleosides, lipids and fatty acids, diols, carbohydrates, aromaticcompounds, vitamins and cofactors, and proteins and enzymes. Theirproduction takes place most expediently on the industrial scale byculturing bacteria which have been developed in order to produce andsecrete large quantities of the particular desired substance. Organismsparticularly suitable for this purpose are coryneform bacteria,gram-positive non-pathogenic bacteria.

It is known that amino acids are prepared by fermentation of strains ofcoryneform bacteria, especially Corynebacterium glutamicum. Because ofthe great importance, continuous work is done on improving theproduction processes. Process improvements may relate to fermentationtechnique measures such as, for example, stirring and oxygen supply, orthe composition of the nutrient media, such as, for example, the sugarconcentration during the fermentation, or the working up to give theproduct, for example by ion exchange chromatography or else spraydrying, or the intrinsic performance properties of the microorganismitself.

Methods of recombinant DNA technology have likewise been employed forsome years for strain improvement of Corynebacterium strains producingfine chemical/proteins, by amplifying individual genes and investigatingthe effect on the production of fine chemicals/proteins.

Other ways for developing a process for producing fine chemicals, aminoacids or proteins, or for increasing or improving the productivity of apre-existing process for producing fine chemicals, amino acids orproteins, are to increase or to alter the expression of one or moregenes, and/or to influence the translation of an mRNA by suitablepolynucleotide sequences. In this connection, influencing may includeincreasing, reducing, or else other parameters of the expression ofgenes, such as chronological expression patterns.

Various constituents of bacterial regulatory sequences are known to theskilled worker. A distinction is made between the binding sites forregulators, also called operators, the binding sites for RNA polymeraseholoenzymes, also called −35 and −10 regions, and the binding site forribosomal 16S RNA, also called ribosome binding site or elseShine-Dalgarno sequence.

The sequence of a ribosome binding site, also called Shine-Dalgarnosequence, means for the purposes of this invention polynucleotidesequences which are located up to 20 bases upstream of the translationinitiation codon.

In the literature (E. coli and S. typhimurium, Neidhardt F. C. 1995 ASMPress) it is reported that both the composition of the polynucleotidesequence of the Shine-Dalgarno sequence, the sequence string of thebases, but also the distance of a polynucleotide sequence present in theShine-Dalgarno sequence from has a considerable influence on thetranslation initiation rate.

Nucleic acid sequences having promoter activity can influence theformation of mRNA in various ways. Promoters whose activities areindependent of the physiological growth phase of the organism are calledconstitutive. Other promoters in turn respond to external chemical andphysical stimuli such as oxygen, metabolites, heat, pH, etc. Others inturn show a strong dependence of their activity in different growthphases. For example, promoters showing a particularly pronouncedactivity during the exponential growth phase of microorganisms, or elseprecisely in the stationary phase of microbial growth, are described inthe literature. Both characteristics of promoters may have a beneficialeffect on productivity for a production of fine chemicals and proteins,depending on the metabolic pathway.

For example, promoters which switch off the expression of a gene duringgrowth, but switch it on after an optimal growth, can be used toregulate a gene which controls the production of a metabolite. Themodified strain then displays the same growth parameters as the startingstrain but produces more product per cell. This type of modification mayincrease both the titer (g of product/liter) and the C yield (g ofproduct/g of C source).

It has already been possible to isolate in Corynebacterium species thosenucleotide sequences which can be used to increase or diminish geneexpression. These regulated promoters may increase or reduce the rate atwhich a gene is transcribed, depending on the internal and/or externalconditions of the cell. In some cases, the presence of a particularfactor, known as inducer, can stimulate the rate of transcription fromthe promoter. Inducers may influence transcription from the promotereither directly or indirectly. Another class of factors, known assuppressors, is able to reduce or else inhibit the transcription fromthe promoter. Like the inducers, the suppressors can also act directlyor indirectly. However, temperature-regulated promoters are also known.Thus, the level of transcription of such promoters can be increased orelse diminished for example by increasing the growth temperature abovethe normal growth temperature of the cell.

A small number of promoters from C. glutamicum have been described todate. The promoter of the malate synthase gene from C. glutamicum wasdescribed in DE 4440118. This promoter was inserted upstream of astructural gene coding for a protein. After transformation of such aconstruct into a coryneform bacterium there is regulation of theexpression of the structural gene downstream of the promoter. Expressionof the structural gene is induced as soon as an appropriate inducer isadded to the medium.

Reinscheid et al., Microbiology 145:503 (1999) described atranscriptional fusion between the pta-ack promoter from C. glutamicumand a reporter gene (chloramphenicol acetyltransferase). Cells of C.glutamicum comprising such a transcriptional fusion exhibited increasedexpression of the reporter gene on growth on acetate-containing medium.By comparison with this, transformed cells which grew on glucose showedno increased expression of this reporter gene.

Pa'tek et al., Microbiology 142:1297 (1996) describe some DNA sequencesfrom C. glutamicum which are able to enhance the expression of areporter gene in C. glutamicum cells. These sequences were comparedtogether in order to define consensus sequences for C. glutamicumpromoters.

Further DNA sequences from C. glutamicum which can be used to regulategene expression have been described in the patent WO 02/40679. Theseisolated polynucleotides represent expression units from Corynebacteriumglutamicum which can be used either to increase or else to reduce geneexpression. This patent additionally describes recombinant plasmids onwhich the expression units from Corynebacterium glutamicum areassociated with heterologous genes. The method described herein, offusing a promoter from Corynebacterium glutamicum with a heterologousgene, can be employed inter alia for regulating the genes of amino acidbiosynthesis.

It is an object of the present invention to provide further promotersand/or expression units with advantageous properties.

We have found that this object is achieved by the use of a nucleic acidhaving promoter activity, comprising

-   -   A) the nucleic acid sequence SEQ. ID. NO. 1 or    -   B) a sequence derived from this sequence by substitution,        insertion or deletion of nucleotides and having an identity of        at least 90% at the nucleic acid level with the sequence SEQ.        ID. NO. 1,        -   or    -   C) a nucleic acid sequence which hybridizes with the nucleic        acid sequence SEQ. ID. NO. 1 under stringent conditions, or    -   D) functionally equivalent fragments of the sequences of A), B)        or C) for the transcription of genes.

“Transcription” means according to the invention the process by which acomplementary RNA molecule is produced starting from a DNA template.Proteins such as RNA polymerase, so-called sigma factors andtranscriptional regulator proteins are involved in this process. Thesynthesized RNA is then used as template in the translation process,which then leads to the biosynthetically active protein.

The formation rate with which a biosynthetically active protein isproduced is a product of the rate of transcription and of translation.Both rates can be influenced according to the invention, and thusinfluence the rate of formation of products in a microorganism.

A “promoter” or a “nucleic acid having promoter activity” meansaccording to the invention a nucleic acid which, in a functional linkageto a nucleic acid to be transcribed, regulates the transcription of thisnucleic acid.

A “functional linkage” means in this connection for example thesequential arrangement of one of the nucleic acids of the inventionhaving promoter activity and a nucleic acid sequence to be transcribedand, where appropriate, further regulatory elements such as, forexample, nucleic acid sequences which ensure the transcription ofnucleic acids, and for example a terminator, in such a way that each ofthe regulatory elements is able to fulfill its function in thetranscription of the nucleic acid sequence. A direct linkage in thechemical sense is not absolutely necessary therefor. Genetic controlsequences, such as, for example, enhancer sequences, are able toexercise their function on the target sequence even from more remotepositions or even from other DNA molecules. Arrangements in which thenucleic acid sequence to be transcribed is positioned behind (i.e. atthe 3′ end) of the promoter sequence of the invention, so that the twosequences are covalently connected together, are preferred. In thisconnection, the distance between the promoter sequence and the nucleicacid sequence to be expressed transgenically is preferably fewer than200 base pairs, particularly preferably less than 100 base pairs, veryparticularly preferably less than 50 base pairs.

“Promoter activity” means according to the invention the quantity of RNAformed by the promoter in a particular time, that is to say thetranscription rate.

“Specific promoter activity” means according to the invention thequantity of RNA formed by the promoter in a particular time for eachpromoter.

The term “wild type” means according to the invention the appropriatestarting microorganism.

Depending on the context, the term “microorganism” means the startingmicroorganism (wild type) or a genetically modified microorganism of theinvention, or both.

Preferably, and especially in cases where the microorganism or the wildtype cannot be unambiguously assigned, “wild type” means for thealteration or causing of the promoter activity or transcription rate,for the alteration of causing of the expression activity or expressionrate and for increasing the content of biosynthetic products in eachcase a reference organism.

In a preferred embodiment, this reference organism is Corynebacteriumglutamicum ATCC 13032.

In a preferred embodiment, the starting microorganisms used are alreadyable to produce the desired fine chemical. Particular preference isgiven in this connection among the particularly preferred microorganismsof bacteria of the genus Corynebacterium and the particularly preferredfine chemicals L-lysine, L-methionine and L-threonine to those startingmicroorganisms already able to produce L-lysine, L-methionine and/orL-threonine. These are particularly preferably corynebacteria in which,for example, the gene coding for an aspartokinase (ask gene) isderegulated or the feedback inhibition is abolished or reduced. Suchbacteria have, for example, a mutation leading to a reduction orabolition of the feedback inhibition, such as, for example, the mutationT311I, in the ask gene.

In the case of a “caused promoter activity” or transcription rate inrelation to a gene compared with the wild type, therefore, compared withthe wild type the formation of an RNA which was not present in this wayin the wild type is caused.

In the case of an altered promoter activity or transcription rate inrelation to a gene compared with the wild type, therefore, compared withthe wild type the quantity of RNA produced in a particular time isaltered.

“Altered” means in this connection preferably increased or reduced.

This can take place for example by increasing or reducing the specificpromoter activity of the endogenous promoter of the invention, forexample by mutating the promoter or by stimulating or inhibiting thepromoter.

A further possibility is to achieve the increased promoter activity ortranscription rate for example by regulating the transcription of genesin the microorganism by nucleic acids of the invention having promoteractivity or by nucleic acids with increased specific promoter activity,where the genes are heterologous in relation to the nucleic acids havingpromoter activity.

The regulation of the transcription of genes in the microorganism bynucleic acids of the invention having promoter activity or by nucleicacids with increased specific promoter activity is preferably achievedby

introducing one or more nucleic acids of the invention having promoteractivity, appropriate with altered specific promoter activity, into thegenome of the microorganism so that transcription of one or moreendogenous genes takes place under the control of the introduced nucleicacid of the invention having promoter activity, appropriate with alteredspecific promoter activity, orintroducing one or more genes into the genome of the microorganism sothat transcription of one or more of the introduced genes takes placeunder the control of the endogenous nucleic acids of the inventionhaving promoter activity, where appropriate with altered specificpromoter activity, orintroducing one or more nucleic acid constructs comprising a nucleicacid of the invention having promoter activity, where appropriate withaltered specific promoter activity, and functionally linked one or morenucleic acids to be transcribed, into the microorganism.

The nucleic acids of the invention having promoter activity comprise

-   -   A) the nucleic acid sequence SEQ. ID. NO. 1 or    -   B) a sequence derived from this sequence by substitution,        insertion or deletion of nucleotides and having an identity of        at least 90% at the nucleic acid level with the sequence SEQ.        ID. NO. 1,        -   or    -   C) a nucleic acid sequence which hybridizes with the nucleic        acid sequence SEQ. ID. NO. 1 under stringent conditions, or    -   D) functionally equivalent fragments of the sequences of A), B)        or C).

The nucleic acid sequence SEQ. ID. NO. 1 represents the promotersequence of superoxide dismutase (Psod) from Corynebacterium glutamicum.SEQ. ID. NO. 1 corresponds to the promoter sequence of the wild type.

The invention additionally relates to nucleic acids having promoteractivity comprising a sequence derived from this sequence bysubstitution, insertion or deletion of nucleotides and having anidentity of at least 90% at the nucleic acid level with the sequenceSEQ. ID. NO. 1.

Further natural examples of the invention for promoters of the inventioncan easily be found for example from various organisms whose genomicsequence is known, by identity comparisons of the nucleic acid sequencesfrom databases with the sequence SEQ ID NO: 1 described above.

Artificial promoter sequences of the invention can easily be foundstarting from the sequence SEQ ID NO: 1 by artificial variation andmutation, for example by substitution, insertion or deletion ofnucleotides.

The term “substitution” means in the description the replacement of oneor more nucleotides by one or more nucleotides. “Deletion” is thereplacement of a nucleotide by a direct linkage. Insertions areinsertions of nucleotides into the nucleic acid sequence, with formalreplacement of a direct linkage by one or more nucleotides.

Identity between two nucleic acids means the identity of the nucleotidesover the complete length of the nucleic acid in each case, in particularthe identity calculated by comparison with the aid of the vector NTISuite 7.1 software from Informax (USA) using the Clustal method (HigginsD G, Sharp P M. Fast and sensitive multiple sequence alignments on amicrocomputer. Comput Appl. Biosci. 1989 April; 5(2):151-1), setting thefollowing parameters:

Multiple Alignment Parameter:

Gap opening penalty 10Gap extension penalty 10Gap separation penalty range 8Gap separation penalty off% identity for alignment delay 40Residue specific gaps offHydrophilic residue gap offTransition weighing 0

Pairwise Alignment Parameter:

FAST algorithm on

K-tuplesize 1

Gap penalty 3Window size 5Number of best diagonals 5

A nucleic acid sequence having an identity of at least 90% with thesequence SEQ ID NO: 1 accordingly means a nucleic acid sequence which,on comparison of its sequence with the sequence SEQ ID NO: 1, inparticular in accordance with the above programming algorithm with theabove parameter set, shows an identity of at least 90%.

Particularly preferred promoters show an identity of 91%, morepreferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, particularly preferably99%, with the nucleic acid sequence SEQ. ID. NO. 1.

Further natural examples of promoters can moreover easily be foundstarting from the nucleic acid sequences described above, in particularstarting from the sequence SEQ ID NO: 1 from various organisms whosegenomic sequence is unknown, by hybridization techniques in a mannerknown per se.

A further aspect of the invention therefore relates to nucleic acidshaving promoter activity comprising a nucleic acid sequence whichhybridizes with the nucleic acid sequence SEQ. ID. No. 1 under stringentconditions. This nucleic acid sequence comprises at least 10, morepreferably more than 12, 15, 30, 50 or particularly preferably more than150, nucleotides.

The hybridization takes place according to the invention under stringentconditions. Such hybridization conditions are described for example inSambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (ALaboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6:

Stringent hybridization conditions mean in particular:

incubation at 42° C. overnight in a solution consisting of 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate and 20g/ml denatured, sheared salmon sperm DNA, followed by washing thefilters with 0.1×SSC at 65° C.

A “functionally equivalent fragment” means for nucleic acid sequenceshaving promoter activity fragments which have substantially the same ora higher specific promoter activity than the starting sequence.

“Essentially identical” means a specific promoter activity whichdisplays at least 50%, preferably 60%, more preferably 70%, morepreferably 80%, more preferably 90%, particularly preferably 95% of thespecific promoter activity of the starting sequence.

“Fragments” mean partial sequences of the nucleic acids having promoteractivity which are described by embodiment A), B) or C). These fragmentspreferably have more than 10, but more preferably more than 12, 15, 30,50 or particularly preferably more than 150, connected nucleotides ofthe nucleic acid sequence SEQ. ID. NO. 1.

It is particularly preferred to use the nucleic acid sequence SEQ. ID.NO. 1 as promoter, i.e. for transcription of genes.

SEQ. ID. NO. 1 has been described without assignment of function in theGenbank entry AP005283. The invention therefore further relates to thenovel nucleic acid sequences of the invention having promoter activity.

The invention relates in particular to a nucleic acid having promoteractivity, comprising

-   -   A) the nucleic acid sequence SEQ. ID. NO 1 or    -   B) a sequence derived from this sequence by substitution,        insertion or deletion of nucleotides and having an identity of        at least 90% at the nucleic acid level with the sequence SEQ.        ID. NO. 1,        -   or    -   C) a nucleic acid sequence which hybridizes with the nucleic        acid sequence SEQ. ID. NO 1 under stringent conditions, or    -   D) functionally equivalent fragments of the sequences of A), B)        or C),        with the proviso that the nucleic acid having the sequence SEQ.        ID. NO. 1 is excluded.

All the nucleic acids having promoter activity which are mentioned abovecan additionally be prepared in a manner known per se by chemicalsynthesis from the nucleotide building blocks such as, for example, byfragment condensation of individual overlapping complementary nucleicacid building blocks of the double helix. The chemical synthesis ofoligonucleotides can take place for example in known manner by thephosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New York,pp. 896-897). Addition of synthetic oligonucleotides and filling in ofgaps using the Klenow fragment of DNA polymerase and ligation reactions,and general cloning methods, are described in Sambrook et al. (1989),Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress.

The invention further relates to the use of an expression unitcomprising one of the nucleic acids of the invention having promoteractivity and additionally functionally linked a nucleic acid sequencewhich ensures the translation of ribonucleic acids for the expression ofgenes.

An expression unit means according to the invention a nucleic acidhaving expression activity, i.e a nucleic acid which, in functionallinkage to a nucleic acid to be expressed, or gene, regulates theexpression, i.e. the transcription and the translation of this nucleicacid or of this gene.

A “functional linkage” means in this connection for example thesequential arrangement of one of the expression units of the inventionand of a nucleic acid sequence which is to be expressed transgenicallyand, where appropriate, further regulatory elements such as, forexample, a terminator in such a way that each of the regulatory elementscan fulfill its function in the transgenic expression of the nucleicacid sequence. A direct linkage in the chemical sense is not absolutelynecessary for this. Genetic control sequences, such as, for example,enhancer sequences, can exercise their function on the target sequencealso from more remote positions or even from different DNA molecules.Arrangements in which the nucleic acid sequence to be expressedtransgenically is positioned behind (i.e. at the 3′ end) the expressionunit sequence of the invention, so that the two sequences are covalentlyconnected together, are preferred. It is preferred in this case for thedistance between the expression unit sequence and the nucleic acidsequence to be expressed transgenically to be less than 200 base pairs,particularly preferably fewer than 100 base pairs, very particularlypreferably fewer than 50 base pairs.

“Expression activity” means according to the invention the quantity ofprotein produced in a particular time by the expression unit, i.e. theexpression rate.

“Specific expression activity” means according to the invention thequantity of protein produced by the expression unit in a particular timefor each expression unit.

In the case of a “caused expression activity” or expression rate inrelation to a gene compared with the wild type, therefore, compared withthe wild type the production of a protein which was not present in thisway in the wild type is caused.

In the case of an “altered expression activity” or expression rate inrelation to a gene compared with the wild type, therefore, compared withthe wild type the quantity of protein produced in a particular time isaltered.

“Altered” preferably means in this connection increased or decreased.

This can take place for example by increasing or reducing the specificactivity of the endogenous expression unit, for example by mutating theexpression unit or by stimulating or inhibiting the expression unit.

The increased expression activity or expression rate can moreover beachieved for example by regulating the expression of genes in themicroorganism by expression units of the invention or by expressionunits with increased specific expression activity, where the genes areheterologous in relation to the expression units.

The regulation of the expression of genes in the microorganism byexpression units of the invention or by expression units of theinvention with increased specific expression activity is preferablyachieved by

introducing one or more expression units of the invention, whereappropriate with altered specific expression activity, into the genomeof the microorganism so that expression of one or more endogenous genestakes place under the control of the introduced expression units of theinvention, where appropriate with altered specific expression activity,orintroducing one or more genes into the genome of the microorganism sothat expression of one or more of the introduced genes takes place underthe control of the endogenous expression units of the invention, whereappropriate with altered specific expression activity, orintroducing one or more nucleic acid constructs comprising an expressionunit of the invention, where appropriate with altered specificexpression activity, and functionally linked one or more nucleic acidsto be expressed, into the microorganism.

The expression units of the invention comprise a nucleic acid of theinvention, described above, having promoter activity and additionallyfunctionally linked a nucleic acid sequence which ensures thetranslation of ribonucleic acids.

This nucleic acid sequence which ensures the translation of ribonucleicacids preferably comprises the nucleic acid sequence SEQ. ID. NO. 42 asribosome binding site.

In a preferred embodiment, the expression unit of the inventioncomprises:

-   -   E) the nucleic acid sequence SEQ. ID. NO. 2 or    -   F) a sequence derived from this sequence by substitution,        insertion or deletion of nucleotides and having an identity of        at least 90% at the nucleic acid level with the sequence SEQ.        ID. NO. 2, or    -   G) a nucleic acid sequence which hybridizes with the nucleic        acid sequence SEQ. ID. NO. 2 under stringent conditions, or    -   H) functionally equivalent fragments of the sequences of E), F)        or G).

The nucleic acid sequence SEQ. ID. NO. 2 represents the nucleic acidsequence of the expression unit of superoxide dismutase (Psod) fromCorynebacterium glutamicum. SEQ. ID. NO. 2 corresponds to the sequenceof the expression unit of the wild type.

The invention further relates to expression units comprising a sequencewhich is derived from this sequence by substitution, insertion ordeletion of nucleotides and which have an identity of at least 90% atthe nucleic acid level with the sequence SEQ. ID. NO. 2.

Further natural examples of the invention for expression units of theinvention can easily be found for example from various organisms whosegenomic sequence is known, by identity comparisons of the nucleic acidsequences from databases with the sequence SEQ ID NO: 2 described above.

Artificial sequences of the invention of the expression units can easilybe found starting from the sequence SEQ ID NO: 2 by artificial variationand mutation, for example by substitution, insertion or deletion ofnucleotides.

A nucleic acid sequence having an identity of at least 90% with thesequence SEQ ID NO: 2 accordingly means a nucleic acid sequence which,on comparison of its sequence with the sequence SEQ ID NO: 2, inparticular in accordance with the above programming algorithm with theabove parameter set, shows an identity of at least 90%.

Particularly preferred expression units show an identity of 91%, morepreferably 92%, 93%, 94%, 95%, 96%, 97%, 98%, particularly preferably99%, with the nucleic acid sequence SEQ. ID. NO. 2.

Further natural examples of expression units can moreover easily befound starting from the nucleic acid sequences described above, inparticular starting from the sequence SEQ ID NO: 2 from variousorganisms whose genomic sequence is unknown, by hybridization techniquesin a manner known per se.

A further aspect of the invention therefore relates to expression unitscomprising a nucleic acid sequence which hybridizes with the nucleicacid sequence SEQ. ID. No. 2 under stringent conditions. This nucleicacid sequence comprises at least 10, more preferably more than 12, 15,30, 50 or particularly preferably more than 150, nucleotides.

“Hybridization” means the ability of a poly- or oligonucleotide to bindunder stringent conditions to a virtually complementary sequence, whilenonspecific bindings between non-complementary partners do not occurunder these conditions. For this, the sequences ought preferably to be90-100% complementary. The property of complementary sequences beingable to bind specifically to one another is made use of for example inthe Northern or Southern blotting technique or in primer binding in PCRor RT-PCR.

The hybridization takes place according to the invention under stringentconditions. Such hybridization conditions are described for example inSambrook, J., Fritsch, E. F., Maniatis, T., in: Molecular Cloning (ALaboratory Manual), 2nd edition, Cold Spring Harbor Laboratory Press,1989, pages 9.31-9.57 or in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6:

Stringent hybridization conditions mean in particular:

incubation at 42° C. overnight in a solution consisting of 50%formamide, 5×SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodiumphosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate and 20g/ml denatured, sheared salmon sperm DNA, followed by washing thefilters with 0.1×SSC at 65° C.

The nucleotide sequences of the invention further make it possible toproduce probes and primers which can be used for identifying and/orcloning homologous sequences in other cell types and microorganisms.Such probes and primers normally comprise a nucleotide sequence regionwhich hybridizes under stringent conditions onto a least approximately12, preferably at least approximately 25, such as, for example,approximately 40, 50 or 75 consecutive nucleotides of a sense strand ofa nucleic acid sequence of the invention or of a corresponding antisensestrand.

Also comprised according to the invention are nucleic acid sequenceswhich comprise so-called silent mutations or are modified in accordancewith the codon usage of a specific original or host organism comparedwith a specifically mentioned sequence, as well as naturally occurringvariants such as, for example, splice variants or allelic variants,thereof.

A “functionally equivalent fragment” means for expression unitsfragments which have substantially the same or a higher specificexpression activity than the starting sequence.

“Essentially identical” means a specific expression activity whichdisplays at least 50%, preferably 60%, more preferably 70%, morepreferably 80%, more preferably 90%, particularly preferably 95% of thespecific expression activity of the starting sequence.

“Fragments” mean partial sequences of the expression units which aredescribed by embodiment E), F) or G). These fragments preferably havemore than 10, but more preferably more than 12, 15, 30, 50 orparticularly preferably more than 150, connected nucleotides of thenucleic acid sequence SEQ. ID. NO 1.

It is particularly preferred to use the nucleic acid sequence SEQ. ID.NO. 2 as expression unit, i.e. for expression of genes.

SEQ. ID. NO. 2 has been described without assignment of function in theGenbank entry AP005283. The invention therefore further relates to thenovel expression units of the invention.

The invention relates in particular to an expression unit comprising anucleic acid of the invention having promoter activity and additionallyfunctionally linked a nucleic acid sequence which ensures thetranslation of ribonucleic acids.

The invention particularly preferably relates to an expression unitcomprising

-   -   E) the nucleic acid sequence SEQ. ID. NO. 2 or    -   F) a sequence derived from this sequence by substitution,        insertion or deletion of nucleotides and having an identity of        at least 90% at the nucleic acid level with the sequence SEQ.        ID. NO. 2, or    -   G) a nucleic acid sequence which hybridizes with the nucleic        acid sequence SEQ. ID. NO. 2 under stringent conditions, or    -   H) functionally equivalent fragments of the sequences of E), F)        or G),        with the proviso that the nucleic acid having the sequence SEQ.        ID. NO. 2 is excluded.

The expression units of the invention comprise one or more of thefollowing genetic elements: a minus 10 (“−10”) sequence; a minus 35(“−35”) sequence; a transcription sequence start, an enhancer region;and an operator region.

These genetic elements are preferably specific for species ofcorynebacteria, especially for Corynbacterium glutamicum.

All the expression units which are mentioned above can additionally beprepared in a manner known per se by chemical synthesis from thenucleotide building blocks such as, for example, by fragmentcondensation of individual overlapping complementary nucleic acidbuilding blocks of the double helix. The chemical synthesis ofoligonucleotides can take place for example in known manner by thephosphoramidite method (Voet, Voet, 2nd edition, Wiley Press New York,pp. 896-897). Addition of synthetic oligonucleotides and filling in ofgaps using the Klenow fragment of DNA polymerase and ligation reactions,and general cloning methods, are described in Sambrook et al. (1989),Molecular cloning: A laboratory manual, Cold Spring Harbor LaboratoryPress.

The methods and techniques used for the inventions in this patent areknown to the skilled worker trained in microbiological and recombinantDNA techniques. Methods and techniques for growing bacterial cells,inserting isolated DNA molecules into the host cell, and isolating,cloning and sequencing isolated nucleic acid molecules etc. are examplesof such techniques and methods. These methods are described in manystandard literature sources: Davis et al., Basic Methods In MolecularBiology (1986); J. H. Miller, Experiments in Molecular Genetics, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1972); J. H.Miller, A Short Course in Bacterial Genetics, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1992); M. Singer and P.Berg, Genes & Genomes, University Science Books, Mill Valley, Calif.(1991); J. Sambrook, E. F. Fritsch and T. Maniatis, Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989); P. B. Kaufmann et al., Handbook of Molecularand Cellular Methods in Biology and Medicine, CRC Press, Boca Raton,Fla. (1995); Methods in Plant Molecular Biology and Biotechnology, B. R.Glick and J. E. Thompson, eds., CRC Press, Boca Raton, Fla. (1993); andP. F. Smith-Keary, Molecular Genetics of Escherichia coli, The GuilfordPress, New York, N.Y. (1989).

All nucleic acid molecules of the present invention are preferably inthe form of an isolated nucleic acid molecule. An “isolated” nucleicacid molecule is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid, and may additionallybe substantially free of other cellular material or culture medium if itis prepared by recombinant techniques, or free of chemical precursors orother chemicals if it is chemically synthesized.

The invention additionally includes the nucleic acid moleculescomplementary to the specifically described nucleotide sequences, or asection thereof.

The promoters and/or expression units of the invention can for examplebe used particularly advantageously in improved methods for thepreparation of biosynthetic products by fermentation as describedhereinafter.

The promoters and/or expression units of the invention have inparticular the advantage that they are induced in microorganisms bystress. It is possible by suitable control of the fermentation processto control this stress induction specifically for an increase in thetranscription/expression rate of desired genes. In the production ofL-lysine in particular, this stress phase is reached very early, so thatin this case an increased transcription/expression rate of desired genescan be achieved very early.

The nucleic acids of the invention having promoter activity can be usedto alter, i.e. to increase or reduce, or to cause the transcription rateof genes in microorganisms compared with the wild type.

The expression units of the invention can be used to alter, i.e. toincrease or reduce, or to cause the expression rate of genes inmicroorganisms compared with the wild type.

The nucleic acids of the invention having promoter activity and theexpression units of the invention can also serve to regulate and enhancethe production of various biosynthetic products such as, for example,fine chemicals, proteins, in particular amino acids, microorganisms, inparticular in Corynebacterium species.

The invention therefore relates to a method for altering or causing thetranscription rate of genes in microorganisms compared with the wildtype by

-   -   a) altering the specific promoter activity in the microorganism        of endogenous nucleic acids of the invention having promoter        activity, which regulate the transcription of endogenous genes,        compared with the wild type or    -   b) regulating transcription of genes in the microorganism by        nucleic acids of the invention having promoter activity or by        nucleic acids with altered specific promoter activity as set        forth in embodiment a), where the genes are heterologous in        relation to the nucleic acids having promoter activity.

According to embodiment a), the alteration or causing of thetranscription rate of genes in the microorganism compared with the wildtype can take place by altering, i.e. increasing or reducing, thespecific promoter activity in the microorganism. This can take place forexample by targeted mutation of the nucleic acid sequence of theinvention having promoter activity, i.e. by targeted substitution,deletion or insertion of nucleotides. An increased or reduced promoteractivity can be achieved by replacing nucleotides in the RNA polymeraseholoenzyme binding sites (known to the skilled worker also as −10 regionand −35 region). Additionally by reducing or enlarging the distance ofthe described RNA polymerase holoenzyme binding sites from one anotherby deleting nucleotides or inserting nucleotides. Additionally byputting binding sites (also known to the skilled worker as operators)for regulatory proteins (known to the skilled worker as repressors andactivators) in the spatial vicinity of the binding sites of the RNApolymerase holoenzyme so that, after binding to a promoter sequence,these regulators diminish or enhance the binding and transcriptionactivity of the RNA polymerase holoenzyme, or else place it under a newregulatory influence.

The nucleic acid sequence SEQ. ID. NO. 44 preferably represents theribosome binding site of the expression units of the invention, and thesequences SEQ. ID. NOs. 42 or 43 represent the −10 region of theexpression units of the invention. Alterations in the nucleic acidsequence in these regions lead to an alteration in the specificexpression activity.

The invention therefore relates to the use of the nucleic acid sequenceSEQ. ID. NO. 44 as ribosome binding site in expression units whichenable genes to be expressed in bacteria of the genus Corynebacterium orBrevibacterium.

The invention further relates to the use of the nucleic acid sequencesSEQ. ID. NOs. 42 or 43 as −10 region in expression units which enablegenes to be expressed in bacteria of the genus Corynebacterium orBrevibacterium.

The invention relates in particular to an expression unit which enablesgenes to be expressed in bacteria of the genus Corynebacterium orBrevibacterium, comprising the nucleic acid sequence SEQ. ID. NO. 44. Inthis case, the nucleic acid sequence SEQ. ID. NO. 44 is preferably usedas ribosome binding site.

The invention further relates to an expression unit which enables genesto be expressed in bacteria of the genus Corynebacterium orBrevibacterium, comprising at least one of the nucleic acid sequencesSEQ. ID. NOs. 42 or 43. In this case, one of the nucleic acid sequencesSEQ. ID. NOs. 42 or 43 is preferably used as −10 region.

In relation to the “specific promoter activity”, an increase orreduction compared with the wild type means an increase or reduction inthe specific activity compared with the nucleic acid of the inventionhaving promoter activity of the wild type, i.e. for example comparedwith SEQ. ID. NO. 1.

According to embodiment b), the alteration or causing of thetranscription rate of genes in microorganisms compared with the wildtype can take place by regulating the transcription of genes in themicroorganism by nucleic acids of the invention having promoter activityor by nucleic acids with altered specific promoter activity according toembodiment a), where the genes are heterologous in relation to thenucleic acids having promoter activity.

This is preferably achieved by

-   -   b1) introducing one or more nucleic acids of the invention        having promoter activity, where appropriate with altered        specific promoter activity, into the genome of the microorganism        so that transcription of one or more endogenous genes takes        place under the control of the introduced nucleic acid having        promoter activity, where appropriate with altered specific        promoter activity, or    -   b2) introducing one or more genes into the genome of the        microorganism so that transcription of one or more of the        introduced genes takes place under the control of the endogenous        nucleic acids of the invention having promoter activity, where        appropriate with altered specific promoter activity, or    -   b3) introducing one or more nucleic acid constructs comprising a        nucleic acid of the invention having promoter activity, where        appropriate with altered specific promoter activity, and        functionally linked one or more nucleic acids to be transcribed,        into the microorganism.

It is thus possible to alter, i.e. to increase or to reduce, thetranscription rate of an endogenous gene of the wild type by

according to embodiment b1), introducing one or more nucleic acids ofthe invention having promoter activity, where appropriate with alteredspecific promoter activity, into the genome of the microorganism so thattranscription of one or more endogenous genes takes place under thecontrol of the introduced nucleic acid having promoter activity, whereappropriate with altered specific promoter activity, oraccording to embodiment b2), introducing one or more endogenous genesinto the genome of the microorganism so that transcription of one ormore of the introduced endogenous genes takes place under the control ofthe endogenous nucleic acids of the invention having promoter activity,where appropriate with altered specific promoter activity, oraccording to embodiment b3), introducing one or more nucleic acidconstructs comprising a nucleic acid of the invention having promoteractivity, where appropriate with altered specific promoter activity, andfunctionally linked one or more endogenous nucleic acids to betranscribed, into the microorganism.

It is thus further possible to cause the transcription rate of anexogenous gene compared with the wild type by

according to embodiment b2), introducing one or more endogenous genesinto the genome of the microorganism so that transcription of one ormore of the introduced exogenous genes takes place under the control ofthe endogenous nucleic acids of the invention having promoter activity,where appropriate with altered specific promoter activity, oraccording to embodiment b3), introducing one or more nucleic acidconstructs comprising a nucleic acid of the invention having promoteractivity, where appropriate with altered specific promoter activity, andfunctionally linked one or more exogenous nucleic acids to betranscribed, into the microorganism.

The insertion of genes according to embodiment b2) can moreover takeplace by integrating a gene into coding regions or noncoding regions.Insertion preferably takes place into noncoding regions.

Insertion of nucleic acid constructs according to embodiment b3) maymoreover take place chromosomally or extrachromosomally. There ispreferably chromosomal insertion of the nucleic acid constructs. A“chromosomal” integration is the insertion of an exogenous DNA fragmentinto the chromosome of a host cell. This term is also used forhomologous recombination between an exogenous DNA fragment and theappropriate region on the chromosome of the host cell.

In embodiment b) there is preferably also use of nucleic acids of theinvention with altered specific promoter activity in accordance withembodiment a). In embodiment b), as described in embodiment a), thesemay be present or be prepared in the microorganism, or be introduced inisolated form into the microorganism.

“Endogenous” means genetic information, such as, for example, genes,which is already present in the wild-type genome.

“Exogenous” means genetic information, such as, for example, genes,which is not present in the wild-type genome.

The term “genes” in relation to regulation of transcription by thenucleic acids of the invention having promoter activity preferably meansnucleic acids which comprise a region to be transcribed, i.e. forexample a region which regulates the translation, and a coding regionand, where appropriate, further regulatory elements such as, forexample, a terminator.

The term “genes” in relation to the regulation, described hereinafter,of expression by the expression units of the invention preferably meansnucleic acids which comprise a coding region and, where appropriate,further regulatory elements such as, for example, a terminator.

A “coding region” means a nucleic acid sequence which encodes a protein.

“Heterologous” in relation to nucleic acids having promoter activity andgenes means that the genes used are not in the wild type transcribedunder the regulation of the nucleic acids of the invention havingpromoter activity, but that a new functional linkage which does notoccur in the wild type is produced, and the functional combination ofnucleic acid of the invention having promoter activity and specific genedoes not occur in the wild type.

“Heterologous” in relation to expression units and genes means that thegenes used are not in the wild type expressed under the regulation ofthe expression units of the invention having promoter activity, but thata new functional linkage which does not occur in the wild type isproduced, and the functional combination of expression unit of theinvention and specific gene does not occur in the wild type.

The invention further relates in a preferred embodiment to a method forincreasing or causing the transcription rate of genes in microorganismscompared with the wild type by

-   -   ah) increasing the specific promoter activity in the        microorganism of endogenous nucleic acids of the invention        having promoter activity, which regulate the transcription of        endogenous genes, compared with the wild type, or    -   bh) regulating the transcription of genes in the microorganism        by nucleic acids of the invention having promoter activity or by        nucleic acids with increased specific promoter activity as set        forth in embodiment a), where the genes are heterologous in        relation to the nucleic acids having promoter activity.

The regulation of the transcription of genes in the microorganism bynucleic acids of the invention having promoter activity or by nucleicacids of the invention with increased specific promoter activityaccording to embodiment ah) is preferably achieved by

-   -   bh1) introducing one or more nucleic acids of the invention        having promoter activity, where appropriate with increased        specific promoter activity, into the genome of the microorganism        so that transcription of one or more endogenous genes takes        place under the control of the introduced nucleic acid of the        invention having promoter activity, where appropriate with        increased specific promoter activity, or    -   bh2) introducing one or more genes into the genome of the        microorganism so that transcription of one or more of the        introduced genes takes place under the control of the endogenous        nucleic acids of the invention having promoter activity, where        appropriate with increased specific promoter activity, or    -   bh3) introducing one or more nucleic acid constructs comprising        a nucleic acid of the invention having promoter activity, where        appropriate with increased specific promoter activity, and        functionally linked one or more nucleic acids to be transcribed,        into the microorganism.

The invention further relates in a preferred embodiment to a method forreducing the transcription rate of genes in microorganisms compared withthe wild type by

-   -   ar) reducing the specific promoter activity in the microorganism        of endogenous nucleic acids of the invention having promoter        activity, which regulate the transcription of the endogenous        genes, compared with the wild type, or    -   br) introducing nucleic acids with reduced specific promoter        activity as set forth in embodiment a) into the genome of the        microorganism so that transcription of endogenous genes takes        place under the control of the introduced nucleic acid with        reduced promoter activity.

The invention further relates to a method for altering or causing theexpression rate of a gene in microorganisms compared with the wild typeby

-   -   c) altering the specific expression activity in the        microorganism of endogenous expression units of the invention,        which regulate the expression of the endogenous genes, compared        with the wild type, or    -   d) regulating the expression of genes in the microorganism by        expression units of the invention or by expression units of the        invention with altered specific expression activity as set forth        in embodiment c), where the genes are heterologous in relation        to the expression units.

According to embodiment c), the alteration or causing of the expressionrate of genes in microorganisms compared with the wild type can takeplace by altering, i.e. increasing or reducing, the specific expressionactivity in the microorganism. This can take place for example bytargeted mutation of the nucleic acid sequence of the invention havingpromoter activity, i.e. by targeted substitution, deletion or insertionof nucleotides. For example, extending the distance betweenShine-Dalgarno sequence and the translation start codon usually leads toa change, a diminution or else an enhancement of the specific expressionactivity. An alteration of the specific expression activity can also beachieved by either shortening or extending the distance of the sequenceof the Shine-Dalgarno region (ribosome binding site) from thetranslation start codon through deletions or insertions of nucleotides.But also by altering the sequence of the Shine-Dalgarno region in such away that the homology to complementary 3′ side 16S rRNA is eitherenhanced or else diminished.

In relation to the “specific expression activity”, an increase orreduction compared with the wild type means an increase or reduction ofthe specific activity compared with the expression unit of the inventionof the wild type, i.e. for example compared with SEQ. ID. NO. 2.

According to embodiment d), the alteration or causing of the expressionrate of genes in microorganisms compared with the wild type can takeplace by regulating the expression of genes in the microorganism byexpression units of the invention or by expression units of theinvention with altered specific expression activity as set forth inembodiment c), where the genes are heterologous in relation to theexpression units.

This is preferably achieved by

-   -   d1) introducing one or more expression units of the invention,        where appropriate with altered specific expression activity,        into the genome of the microorganism so that expression of one        or more endogenous genes takes place under the control of the        introduced expression units, or    -   d2) introducing one or more genes into the genome of the        microorganism so that expression of one or more of the        introduced genes takes place under the control of the endogenous        expression units of the invention, where appropriate with        altered specific expression activity, or    -   d3) introducing one or more nucleic acid constructs comprising        an expression unit of the invention, where appropriate with        altered specific expression activity, and functionally linked        one or more nucleic acids to be expressed, into the        microorganism.

It is thus possible to alter, i.e. to increase or to reduce, theexpression rate of an endogenous gene of the wild type by

according to embodiment d1) introducing one or more expression units ofthe invention, where appropriate with altered specific expressionactivity, into the genome of the microorganism so that expression of oneor more endogenous genes takes place under the control of the introducedexpression units, oraccording to embodiment d2) introducing one or more genes into thegenome of the microorganism so that expression of one or more of theintroduced genes takes place under the control of the endogenousexpression units of the invention, whereappropriate with altered specific expression activity, or according toembodiment d3) introducing one or more nucleic acid constructscomprising an expression unit of the invention, where appropriate withaltered specific expression activity, and functionally linked one ormore nucleic acids to be expressed, into the microorganism.

It is thus further possible to cause the expression rate of anendogenous gene compared with the wild type by

according to embodiment d2) introducing one or more exogenous genes intothe genome of the microorganism so that expression of one or more of theintroduced genes takes place under the control of the endogenousexpression units of the invention, where appropriate with alteredspecific expression activity, oraccording to embodiment d3) introducing one or more nucleic acidconstructs comprising an expression unit of the invention, whereappropriate with altered specific expression activity, and functionallylinked one or more exogenous nucleic acids to be expressed, into themicroorganism.

The insertion of genes according to embodiment d2) can moreover takeplace by integrating a gene into coding regions or noncoding regions.Insertion preferably takes place into noncoding regions.

Insertion of nucleic acid constructs according to embodiment d3) maymoreover take place chromosomally or extrachromosomally. There ispreferably chromosomal insertion of the nucleic acid constructs.

The nucleic acid constructs are also referred to hereinafter asexpression cassettes.

In embodiment d) there is preferably also use of expression units of theinvention with altered specific expression activity in accordance withembodiment c). In embodiment d), as described in embodiment c), thesemay be present or be prepared in the microorganism, or be introduced inisolated form into the microorganism.

The invention further relates in a preferred embodiment to a method forincreasing or causing the expression rate of a gene in microorganismscompared with the wild type by

ch) increasing the specific expression activity in the microorganism ofendogenous expression units of the invention, which regulate theexpression of the endogenous genes, compared with the wild type, ordh) regulating the expression of genes in the microorganism byexpression units of the invention or by expression units with increasedspecific expression activity as set forth in embodiment a), where thegenes are heterologous in relation to the expression units.

The regulation of the expression of genes in the microorganism byexpression units of the invention or by expression units with increasedspecific expression activity as set forth in embodiment c) is preferablyachieved by

-   -   dh1) introducing one or more expression units of the invention,        where appropriate with increased specific expression activity,        into the genome of the microorganism so that expression of one        or more endogenous genes takes place under the control of the        introduced expression units, where appropriate with increased        specific expression activity, or    -   dh2) introducing one or more genes into the genome of the        microorganism so that expression of one or more of the        introduced genes takes place under the control of the endogenous        expression units of the invention, where appropriate with        increased specific expression activity, or    -   dh3) introducing one or more nucleic acid constructs comprising        an expression unit of the invention, where appropriate with        increased specific expression activity, and functionally linked        one or more nucleic acids to be expressed, into the        microorganism.

The invention further relates to a method for reducing the expressionrate of genes in microorganisms compared with the wild type by

cr) reducing the specific expression activity in the microorganism ofendogenous expression units of the invention, which regulate theexpression of the endogenous genes, compared with the wild type, ordr) introducing expression units with reduced specific expressionactivity as set forth in embodiment cr) into the genome of themicroorganism so that expression of endogenous genes takes place underthe control of the introduced expression units with reduced expressionactivity.

In a preferred embodiment of the methods of the invention describedabove for altering or causing the transcription rate and/or expressionrate of genes in microorganisms, the genes are selected from the groupof nucleic acids encoding a protein from the biosynthetic pathway offine chemicals, where the genes may optionally comprise furtherregulatory elements.

In a particularly preferred embodiment of the methods of the inventiondescribed above for altering or causing the transcription rate and/orexpression rate of genes in microorganisms, the genes are selected fromthe group of nucleic acids encoding a protein from the biosyntheticpathway of proteinogenic and non-proteinogenic amino acids, nucleicacids encoding a protein from the biosynthetic pathway of nucleotidesand nucleosides, nucleic acids encoding a protein from the biosyntheticpathway of organic acids, nucleic acids encoding a protein from thebiosynthetic pathway of lipids and fatty acids, nucleic acids encoding aprotein from the biosynthetic pathway of diols, nucleic acids encoding aprotein from the biosynthetic pathway of carbohydrates, nucleic acidsencoding a protein from the biosynthetic pathway of aromatic compounds,nucleic acids encoding a protein from the biosynthetic pathway ofvitamins, nucleic acids encoding a protein from the biosynthetic pathwayof cofactors and nucleic acids encoding a protein from the biosyntheticpathway of enzymes, where the genes may optionally comprise furtherregulatory elements.

In a particularly preferred embodiment, the proteins from thebiosynthetic pathway of amino acids are selected from the group ofaspartate kinase, aspartate-semialdehyde dehydrogenase, diaminopimelatedehydrogenase, diaminopimelate decarboxylase, dihydrodipicolinatesynthetase, dihydrodipicolinate reductase, glyceraldehyde-3-phosphatedehydrogenase, 3-phosphoglycerate kinase, pyruvate carboxylase,triosephosphate isomerase, transcriptional regulator LuxR,transcriptional regulator LysR1, transcriptional regulator LysR2,malate-quinone oxidoreductase, glucose-6-phosphate deydrogenase,6-phosphogluconate dehydrogenase, transketolase, transaldolase,homoserine O-acetyltransferase, cystathionine gamma-synthase,cystathionine beta-lyase, serine hydroxymethyltransferase,O-acetylhomoserine sulfhydrylase, methylenetetrahydrofolate reductase,phosphoserine aminotransferase, phosphoserine phosphatase, serineacetyltransferase, homoserine dehydrogenase, homoserine kinase,threonine synthase, threonine exporter carrier, threonine dehydratase,pyruvate oxidase, lysine exporter, biotin ligase, cysteine synthase I,cysteine synthase II, coenzyme B12-dependent methionine synthase,coenzyme B12-independent methionine synthase activity, sulfateadenylyltransferase subunit 1 and 2, phosphoadenosine-phosphosulfatereductase, ferredoxin-sulfite reductase, ferredoxin NADP reductase,3-phosphoglycerate dehydrogenase, RXA00655 regulator, RXN2910 regulator,arginyl-tRNA synthetase, phosphoenolpyruvate carboxylase, threonineefflux protein, serine hydroxymethyltransferase,fructose-1,6-bisphosphatase, protein of sulfate reduction RXA077,protein of sulfate reduction RXA248, protein of sulfate reductionRXA247, protein OpcA, 1-phosphofructokinase and 6-phosphofructokinase.

Preferred proteins and nucleic acids encoding these proteins of theproteins described above from the biosynthetic pathway of amino acidsare respectively protein sequences and nucleic acid sequences ofmicrobial origin, preferably from bacteria of the genus Corynebacteriumor Brevibacterium, preferably from coryneform bacteria, particularlypreferably from Corynebacterium glutamicum.

Examples of particularly preferred protein sequences and thecorresponding nucleic acid sequences encoding these proteins from thebiosynthetic pathway of amino acids, the document referring thereto, andthe designation thereof in the referring document are listed in Table 1:

TABLE 1 Nucleic SEQ. ID. acid NO. in encoding Referring referringProtein protein document document Aspartate kinase ask or EP1108790 DNA:281 lysC Protein: 3781 Aspartate- asd EP1108790 DNA: 331 semialdehydeProtein: 3831 dehydrogenase Dihydrodipicolinate dapA WO 0100843 DNA: 55synthetase Protein: 56 Dihydrodipicolinate dapB WO 0100843 DNA: 35reductase Protein: 36 meso-Diaminopime- ddh EP1108790 DNA: 3494 lateD-dehydrogenase Protein: 6944 Diaminopicolinate lysA EP1108790 DNA: 3451decarboxylase Prot.: 6951 Lysine exporter lysE EP1108790 DNA: 3455Prot.: 6955 Arginyl-tRNA argS EP1108790 DNA: 3450 synthetase Prot.: 6950Glucose-6-phosphate zwf WO 0100844 DNA: 243 dehydrognease Prot.: 244Glyceraldehyde-3- gap WO 0100844 DNA: 187 phosphate Prot.: 188dehydrogenase 3-Phosphoglycerate pgk WO 0100844 DNA: 69 kinase Prot.: 70Pyruvate pycA EP1108790 DNA: 765 carboxylase Prot.: 4265 Triosephosphatetpi WO 0100844 DNA: 61 isomerase Prot.: 62 Biotin ligase birA EP1108790DNA: 786 Prot.: 4286 PEP carboxylase pck EP1108790 DNA: 3470 Prot.: 6970Homoserine kinase thrB WO 0100843 DNA: 173 Prot.: 174 Threonine synthasethrC WO 0100843 DNA: 175 Prot.: 176 Threonine export thrE WO 0251231DNA: 41 carrier Prot.: 42 Threonine efflux RXA2390 WO 0100843 DNA: 7protein Prot.: 8 Threonine ilvA EP 1108790 DNA: 2328 dehydratase Prot.:5828 Homoserine metA EP 1108790 DNA: 727 O-acetyltransferase Prot: 4227Cystathionine gamma- metB EP 1108790 DNA: 3491 synthase Prot: 6991Cystathionine beta- metC EP 1108790 DNA: 2535 lyase Prot: 6035 CoenzymeB12- metH EP 1108790 DNA: 1663 dependent methionine Prot: 5163synthase, - O-Acetylhomoserine metY EP 1108790 DNA: 726 sulfhydrylaseProt: 4226 Methylenetetrahydro- metF EP 1108790 DNA: 2379 folatereductase Prot: 5879 D-3-Phosphoglycerate serA EP 1108790 DNA: 1415dehydrogenase Prot: 4915 Phosphoserine serB WO 0100843 DNA: 153phosphatase 1 Prot.: 154 Phosphoserine serB EP 1108790 DNA: 467phosphatase 2 Prot: 3967 Phosphoserine serB EP 1108790 DNA: 334phosphatase 3 Prot.: 3834 Phosphoserine serC WO 0100843 DNA: 151aminotransferase Prot.: 152 Serine acetyltrans- cysE WO 0100843 DNA: 243ferase Prot.: 244 Cysteine synthase I cysK EP 1108790 DNA: 2817 Prot.:6317 Cysteine synthase II CysM EP 1108790 DNA: 2338 Prot.: 5838Homoserine hom EP 1108790 DNA: 3452 dehydrogenase Prot.: 6952 CoenzymeB12- metE WO 0100843 DNA: 755 independent Prot.: 756 methionine synthaseSerine glyA WO 0100843 DNA: 143 hydroxymethyltrans- Prot.: 144 feraseProtein in sulfate RXA247 EP 1108790 DNA: 3089 reduction Prot.: 6589Protein in sulfate RXA248 EP 1108790 DNA: 3090 reduction Prot.: 6590Sulfate CysN EP 1108790 DNA: 3092 adenylyltransferase Prot.: 6592subunit 1 Sulfate CysD EP 1108790 DNA: 3093 adenylyltransferase Prot.:6593 subunit 2 Phosphoadenosine- CysH WO 02729029 DNA: 7 phosphosulfateProt.: 8 reductase Ferredoxin-sulfite RXA073 WO 0100842 DNA: 329reductase Prot.: 330 Ferredoxin NADP- RXA076 WO 0100843 DNA: 79reductase Prot.: 80 Transcriptional luxR WO 0100842 DNA: 297 regulatorLuxR Protein: 298 Transcriptional lysR1 EP 1108790 DNA: 676 regulatorLysR1 Protein: 4176 Transcriptional lysR2 EP 1108790 DNA: 3228 regulatorLysR2 Protein: 6728 Transcriptional lysR3 EP 1108790 DNA: 2200 regulatorLysR3 Protein: 5700 Malate-quinone mqo WO 0100844 DNA: 569oxidoreductase Protein: 570 Transketolase RXA2739 EP 1108790 DNA: 1740Prot: 5240 Transaldolase RXA2738 WO 0100844 DNA: 245 Prot: 246 OpcA opcAWO 0100804 DNA: 79 Prot: 80 1-Phosphofructo- pfk1 WO 0100844 DNA: 55kinase 1 Protein: 56 1-Phosphofructo- pfk2 WO 0100844 DNA: 57 kinase 2Protein: 58 6-Phosphofructo- 6-pfk1 EP 1108790 DNA: 1383 kinase 1Protein: 4883 6-Phosphofructo- 6-pfk2 DE 10112992 DNA: 1 kinase 2Protein: 2 Fructose-1,6- fbr1 EP1108790 DNA: 1136 bisphosphatase 1Protein: 4636 Pyruvate oxidase poxB WO 0100844 DNA: 85 Protein: 86RXA00655 regulator RXA655 US2003162267.2 DNA: 1 Prot.: 2 RXN02910regulator RXN2910 US2003162267.2 DNA: 5 Prot.: 6 6-phosphoglucono-RXA2735 WO 0100844 DNA: 1 lactonase Prot.: 2

A further example of a particularly preferred protein sequence and thecorresponding nucleic acid sequence encoding this protein from thebiosynthetic pathway of amino acids is the sequence offructose-1,6-bisphosphatase 2, also called fbr2, (SEQ. ID. NO. 41) andthe corresponding nucleic acid sequence encoding afructose-1,6-bisphosphatase 2 (SEQ. ID. NO. 40).

A further example of a particularly preferred protein sequence and thecorresponding nucleic acid sequence encoding this protein from thebiosynthetic pathway of amino acids is the sequence of the protein insulfate reduction, also called RXA077, (SEQ. ID. NO. 4) and thecorresponding nucleic acid sequence encoding a protein in sulfatereduction (SEQ. ID. NO. 3).

Further particularly preferred protein sequences from the biosyntheticpathway of amino acids have in each case the amino acid sequenceindicated in Table 1 for this protein, where the respective protein has,in at least one of the amino acid positions indicated in Table 2/column2 for this amino acid sequence, a different proteinogenic amino acidthan the respective amino acid indicated in Table 2/column 3 in the sameline. In a further preferred embodiment, the proteins have, in at leastone of the amino acid positions indicated in Table 2/column 2 for theamino acid sequence, the amino acid indicated in Table 2/column 4 in thesame line. The proteins indicated in Table 2 are mutated proteins of thebiosynthetic pathway of amino acids, which have particularlyadvantageous properties and are therefore particularly suitable forexpressing the corresponding nucleic acids through the promoter of theinvention and for producing amino acids. For example, the mutation T311Ileads to the feedback inhibition of ask being switched off.

The corresponding nucleic acids which encode a mutated protein describedabove from Table 2 can be prepared by conventional methods.

A suitable starting point for preparing the nucleic acid sequencesencoding a mutated protein is, for example, the genome of aCorynebacterium glutamicum strain which is obtainable from the AmericanType Culture Collection under the designation ATCC 13032, or the nucleicacid sequences referred to in Table 1. For the back-translation of theamino acid sequence of the mutated proteins into the nucleic acidsequences encoding these proteins, it is advantageous to use the codonusage of the organism into which the nucleic acid sequence is to beintroduced or in which the nucleic acid sequence is present. Forexample, it is advantageous to use the codon usage of Corynebacteriumglutamicum for Corynebacterium glutamicum. The codon usage of theparticular organism can be ascertained in a manner known per se fromdatabases or patent applications which describe at least one protein andone gene which encodes this protein from the desired organism.

The information in Table 2 is to be understood in the following way:

In column 1 “identification”, an unambiguous designation for eachsequence in relation to Table 1 is indicated.

In column 2 “AA-POS”, the respective number refers to the amino acidposition of the corresponding polypeptide sequence from Table 1. A “26”in the column “AA-POS” accordingly means amino acid position 26 of thecorrespondingly indicated polypeptide sequence. The numbering of theposition starts at +1 at the N terminus.

In column 3 “AA wild type”, the respective letter designates the aminoacid—represented in one-letter code—at the position indicated in column2 in the corresponding wild-type strain of the sequence from Table 1.

In column 4 “AA mutant”, the respective letter designates the aminoacid—represented in one-letter code—at the position indicated in column2 in the corresponding mutant strain.

In column 5 “function”, the physiological function of the correspondingpolypeptide sequence is indicated.

For mutated protein with a particular function (column 5) and aparticular initial amino acid sequence (Table 1), columns 2, 3 and 4describe at least one mutation, and a plurality of mutations for somesequences. This plurality of mutations always refers to the closestinitial amino acid sequence above in each case (Table 1). The term “atleast one of the amino acid positions” of a particular amino acidsequence preferably means at least one of the mutations described forthis amino acid sequence in columns 2, 3 and 4.

One-letter code for proteinogenic amino acids:

A alanineC cysteineD aspartateE glutamateF phenylalanineG glycineH histidineI isoleucineK lysineL leucineM methionineN asparagineP prolineQ glutamineR arginineS serineT threonineV valineW tryptophanY tyrosine

TABLE 2 Column 1 Column 2 Column 3 Column 4 Identi- AA AA wild AA Column5 fication position type mutant Function ask 317 S A aspartate kinase311 T I 279 A T asd 66 D G aspartate- semialdehyde dehydrogenase 234 R H272 D E 285 K E 20 L F dapA 2 S A dihydrodi- picolinate synthetase 84 KN 85 L V dapB 91 D A dihydrodi- picolinate reductase 83 D N ddh 174 D Emeso- diaminopimelate D-dehydrogenase 235 F L 237 S A lysA 265 A Ddiaminopicolinate decarboxylase 320 D N 332 I V argS 355 G Darginyl-tRNA synthetase 156 A S 513 V A 540 H R zwf 8 S T glucose-6-phosphate dehydrogenase 150 T A 321 G S gap 264 G S glyceraldehyde-3-phosphate dehydrogenase pycA 7 S L pyruvate carboxylase 153 E D 182 AS 206 A S 227 H R 455 A G 458 P S 639 S T 1008 R H 1059 S P 1120 D E pck162 H Y PEP carboxylase 241 G D 829 T R thrB 103 S A homoserine kinase190 T A 133 A V 138 P S thrC 69 G R threonine synthase 478 T I RXA330 85I M threonine efflux protein 161 F I 195 G D hom 104 V I homoserinedehydrogenase 116 T I 148 G A 59 V A 270 T S 345 R P 268 K N 61 D H 72 EQ lysR1 80 R H transcriptional regulator LysR1 lysR3 142 R Wtranscriptional regulator LysR3 179 A T RXA2739 75 N D transketolase 329A T 332 A T 556 V I RXA2738 242 K M transaldolase opcA 107 Y H OpcA 219K N 233 P S 261 Y H 312 S F 65 G R aspartate-1- decarboxylase 33 G S6-phosphoglu- conolactonase

In the methods of the invention described above for altering or causingthe transcription rate and/or expression rate of genes inmicroorganisms, and the methods described hereinafter for producinggenetically modified microorganisms, the genetically modifiedmicroorganisms described hereinafter and the methods describedhereinafter for producing biosynthetic products, the introduction of thenucleic acids of the invention having promoter activity, of theexpression units of the invention, of the genes described above and ofthe nucleic acid constructs or expression cassettes described above intothe microorganism, in particular into coryneform bacteria, preferablytakes place by the SacB method.

The SacB method is known to the skilled worker and described for examplein Schäfer A, Tauch A, Jäger W, Kalinowski J, Thierbach G, Pühler A.;Small mobilizable multi-purpose cloning vectors derived from theEscherichia coli plasmids pK18 and pK19: selection of defined deletionsin the chromosome of Corynebacterium glutamicum, Gene. 1994 Jul. 22;145(1):69-73 and Blomfield I C, Vaughn V, Rest R F, Eisenstein B I.;Allelic exchange in Escherichia coli using the Bacillus subtilis sacBgene and a temperature-sensitive pSC101 replicon; Mol. Microbiol. 1991June; 5(6):1447-57.

In a preferred embodiment of the methods of the invention describedabove, the alteration or causing of the transcription rate and/orexpression rate of genes in microorganisms takes place by introducingnucleic acids of the invention having promoter activity or expressionunits of the invention into the microorganism.

In a further preferred embodiment of the methods of the inventiondescribed above, the alteration or causing of the transcription rateand/or expression rate of genes in microorganisms takes place byintroducing the nucleic acid constructs or expression cassettesdescribed above into the microorganism.

The invention therefore also relates to an expression cassettecomprising

at least one expression unit of the inventionat least one further nucleic acid sequence to be expressed, i.e. a geneto be expressed andwhere appropriate further genetic control elements such as, for example,a terminator, where at least one expression unit and a further nucleicacid sequence to be expressed are functionally linked together, and thefurther nucleic acid sequence to be expressed is heterologous inrelation to the expression unit.

The nucleic acid sequence to be expressed is preferably at least onenucleic acid encoding a protein from the biosynthesis pathway of finechemicals.

The nucleic acid sequence to be expressed is particularly preferablyselected from the group of nucleic acids encoding a protein from thebiosynthetic pathway of proteinogenic and non-proteinogenic amino acids,nucleic acids encoding a protein from the biosynthetic pathway ofnucleotides and nucleosides, nucleic acids encoding a protein from thebiosynthetic pathway of organic acids, nucleic acids encoding a proteinfrom the biosynthetic pathway of lipids and fatty acids, nucleic acidsencoding a protein from the biosynthetic pathway of diols, nucleic acidsencoding a protein from the biosynthetic pathway of carbohydrates,nucleic acids encoding a protein from the biosynthetic pathway ofaromatic compounds, nucleic acids encoding a protein from thebiosynthetic pathway of vitamins, nucleic acids encoding a protein fromthe biosynthetic pathway of cofactors and nucleic acids encoding aprotein from the biosynthetic pathway of enzymes.

Preferred proteins from the biosynthetic pathway of amino acids aredescribed above and examples thereof are described in Tables 1 and 2.

The physical location of the expression unit relative to the gene to beexpressed in the expression cassettes of the invention is chosen so thatthe expression unit regulates the transcription and preferably also thetranslation of the gene to be expressed, and thus enables one or moreproteins to be produced. “Enabling production” includes in thisconnection a constitutive increase in the production, diminution orblocking of production under specific conditions and/or increasing theproduction under specific conditions. The “conditions” comprise in thisconnection: (1) addition of a component to the culture medium, (2)removal of a component from the culture medium, (3) replacement of onecomponent in the culture medium by a second component, (4) increasingthe temperature of the culture medium, (5) reducing the temperature ofthe culture medium, and (6) regulating the atmospheric conditions suchas, for example, the oxygen or nitrogen concentration in which theculture medium is kept.

The invention further relates to an expression vector comprising anexpression cassette of the invention described above.

Vectors are well known to the skilled worker and can be found in“Cloning Vectors” (Pouwels P. H. et al., editors, Elsevier,Amsterdam-New York-Oxford, 1985). Apart from plasmids, vectors also meanall other vectors known to the skilled worker, such as, for example,phages, transposons, IS elements, phasmids, cosmids, and linear orcircular DNA. These vectors may undergo autonomous replication in thehost organism or chromosomal replication.

Suitable and particularly preferred plasmids are those which arereplicated in coryneform bacteria. Numerous known plasmid vectors suchas, for example, pZ1 (Menkel et al., Applied and EnvironmentalMicrobiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107: 69-74 (1991)) arebased on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmidvectors such as, for example, pCLiK5MCS, or those based on pCG4 (U.S.Pat. No. 4,489,160) or pNG2 (Serwold-Davis et al., FEMS MicrobiologyLetters 66, 119-124 (1990)) or pAG1 (U.S. Pat. No. 5,158,891), can beused in the same way.

Also suitable are those plasmid vectors with the aid of which the methodof gene amplification by integration into the chromosome can be used, asdescribed for example by Reinscheid et al. (Applied and EnvironmentalMicrobiology 60, 126-132 (1994)) for the duplication and amplificationof the hom-thrB operon. In this method the complete gene is cloned intoa plasmid vector which is able to replicate in a host (typically E.coli) but not in C. glutamicum. Examples of suitable vectors are pSUP301(Simon et al., Bio/ Technology 1, 784-791 (1983)), pK18mob or pK19mob(Schäfer et al., Gene 145, 69-73 (1994)), Bernard et al., Journal ofMolecular Biology, 234: 534-541 (1993)), pEM1 (Schrumpf et al. 1991,Journal of Bacteriology 173: 4510-4516) or pBGS8 (Spratt et al., 1986,Gene 41: 337-342). The plasmid vector which comprises the gene to beamplified is subsequently transferred by transformation into the desiredstrain of C. glutamicum. Methods for transformation are described forexample in Thierbach et al. (Applied Microbiology and Biotechnology 29,356-362 (1988)), Dunican and Shivnan (Biotechnology 7, 1067-1070 (1989))and Tauch et al. (FEMS Microbiological Letters 123, 343-347 (1994)).

The invention further relates to a genetically modified microorganismwhere the genetic modification leads to an alteration or causing of thetranscription rate of at least one gene compared with the wild type, andis dependent on

a) altering the specific promoter activity in the microorganism of atleast one endogenous nucleic acid having promoter activity as set forthin claim 1, which regulates the transcription of at least one endogenousgene, orb) regulating the transcription of genes in the microorganism by nucleicacids having promoter activity as set forth in claim 1 or by nucleicacids having promoter activity as set forth in claim 1 with alteredspecific promoter activity as set forth in embodiment a), where thegenes are heterologous in relation to the nucleic acids having promoteractivity.

As described above for the methods, the regulation of the transcriptionof genes in the microorganism by nucleic acids having promoter activityas set forth in claim 1 or by nucleic acids having promoter activity asset forth in claim 1 with altered specific promoter activity as setforth in embodiment a), is achieved by

b1) introducing one or more nucleic acids having promoter activity asset forth in claim 1, where appropriate with altered specific promoteractivity, into the genome of the microorganism so that transcription ofone or more endogenous genes takes place under the control of theintroduced nucleic acid having promoter activity as set forth in claim1, where appropriate with altered specific promoter activity, orb2) introducing one or more genes into the genome of the microorganismso that transcription of one or more of the introduced genes takes placeunder the control of the endogenous nucleic acids having promoteractivity as set forth in claim 1, where appropriate with alteredspecific promoter activity, orb3) introducing one or more nucleic acid constructs comprising a nucleicacid having promoter activity as set forth in claim 1, where appropriatewith altered specific promoter activity, and functionally linked one ormore nucleic acids to be transcribed, into the microorganism.

The invention further relates to a genetically modified microorganismhaving elevated or caused transcription rate of at least one genecompared with the wild type, where

ah) the specific promoter activity in the microorganism of endogenousnucleic acids having promoter activity as set forth in claim 1, whichregulate the transcription of endogenous genes, is increased comparedwith the wild type, orbh) the transcription of genes in the microorganism is regulated bynucleic acids having promoter activity as set forth in claim 1 or bynucleic acids having increased specific promoter activity as set forthin embodiment ah), where the genes are heterologous in relation to thenucleic acids having promoter activity.

As described above for the methods, the regulation of the transcriptionof genes in the microorganism by nucleic acids having promoter activityas set forth in claim 1 or by nucleic acids having promoter activity asset forth in claim 1 with increased specific promoter activity as setforth in embodiment a), is achieved by

bh1) introducing one or more nucleic acids having promoter activity asset forth in claim 1, where appropriate with increased specific promoteractivity, into the genome of the microorganism so that transcription ofone or more endogenous genes takes place under the control of theintroduced nucleic acid having promoter activity, where appropriate withincreased specific promoter activity, orbh2) introducing one or more genes into the genome of the microorganismso that transcription of one or more of the introduced genes takes placeunder the control of the endogenous nucleic acids having promoteractivity as set forth in claim 1, where appropriate with increasedspecific promoter activity, orbh3) introducing one or more nucleic acid constructs comprising anucleic acid having promoter activity as set forth in claim 1, whereappropriate with increased specific promoter activity, and functionallylinked one or more nucleic acids to be transcribed, into themicroorganism.

The invention further relates to a genetically modified microorganismwith reduced transcription rate of at least one gene compared with thewild type, where

ar) the specific promoter activity in the microorganism of at least oneendogenous nucleic acid having promoter activity as set forth in claim1, which regulates the transcription of at least one endogenous gene, isreduced compared with the wild type, orbr) one or more nucleic acids having reduced promoter activity as setforth in embodiment a) are introduced into the genome of themicroorganism so that the transcription of at least one endogenous genetakes place under the control of the introduced nucleic acid havingreduced promoter activity.

The invention further relates to a genetically modified microorganism,where the genetic modification leads to an alteration or causing of theexpression rate of at least one gene compared with the wild type, and isdependent on

c) altering the specific expression activity in the microorganism of atleast one endogenous expression unit as set forth in claim 2 or 3, whichregulates the expression of at least one endogenous gene, compared withthe wild type ord) regulating the expression of genes in the microorganism by expressionunits as set forth in claim 2 or 3 or by expression units as set forthin claim 2 or 3 with altered specific expression activity as set forthin embodiment a), where the genes are heterologous in relation to theexpression units.

As described above for the methods, the regulation of the expression ofgenes in the microorganism by expression units as set forth in claim 2or 3 or by expression units as set forth in claim 2 or 3 with alteredspecific expression activity as set forth in embodiment a) is achievedby

d1) introducing one or more expression units as set forth in claim 2 or3, where appropriate with altered specific expression activity, into thegenome of the microorganism so that expression of one or more endogenousgenes takes place under the control of the introduced expression unitsaccording to claim 2 or 3, where appropriate with altered specificexpression activity, ord2) introducing one or more genes into the genome of the microorganismso that expression of one or more of the introduced genes takes placeunder the control of the endogenous expression units as set forth inclaim 2 or 3, where appropriate with altered specific expressionactivity, ord3) introducing one or more nucleic acid constructs comprising anexpression unit as set forth in claim 2 or 3, where appropriate withaltered specific expression activity, and functionally linked one ormore nucleic acids to be expressed, into the microorganism.

The invention further relates to a genetically modified microorganismwith increased or caused expression rate of at least one gene comparedwith the wild type, where

ch) the specific expression activity in the microorganism of at leastone endogenous expression unit as set forth in claim 2 or 3, whichregulates the expression of the endogenous genes, is increased comparedwith the wild type, ordh) the expression of genes in the microorganism is regulated byexpression units as set forth in claim 2 or 3 or by expression units asset forth in claim 2 or 3 with increased specific expression activity asset forth in embodiment a), where the genes are heterologous in relationto the expression units.

As described above for the methods, the regulation of the expression ofgenes in the microorganism by expression units as set forth in claim 2or 3 or by expression units as set forth in claim 2 or 3 with increasedspecific expression activity as set forth in embodiment a) is achievedby

dh1) introducing one or more expression units as set forth in claim 2 or3, where appropriate with increased specific expression activity, intothe genome of the microorganism so that expression of one or moreendogenous genes takes place under the control of the introducedexpression units according to claim 2 or 3, where appropriate withincreased specific expression activity, ordh2) introducing one or more genes into the genome of the microorganismso that expression of one or more of the introduced genes takes placeunder the control of the endogenous expression units as set forth inclaim 2 or 3, where appropriate with increased specific expressionactivity, ordh3) introducing one or more nucleic acid constructs comprising anexpression unit as set forth in claim 2 or 3, where appropriate withincreased specific expression activity, and functionally linked one ormore nucleic acids to be expressed, into the microorganism.

The invention further relates to a genetically modified microorganismwith reduced expression rate of at least one gene compared with the wildtype, where

cr) the specific expression activity in the microorganism of at leastone endogenous expression unit as set forth in claim 2 or 3, whichregulates the expression of at least one endogenous gene, is reducedcompared with the wild type, ordr) one or more expression units according to claim 2 or 3 with reducedexpression activity are introduced into the genome of the microorganismso that expression of at least one endogenous gene takes place under thecontrol of the introduced expression unit according to claim 2 or 3 withreduced expression activity.

The invention further relates to a genetically modified microorganismcomprising an expression unit as set forth in claim 2 or 3 andfunctionally linked a gene to be expressed, where the gene isheterologous in relation to the expression unit.

This genetically modified microorganism particularly preferablycomprises an expression cassette of the invention.

The present invention particularly preferably relates to geneticallymodified microorganisms, in particular coryneform bacteria, whichcomprise a vector, in particular shuttle vector or plasmid vector, whichharbors at least one recombinant nucleic acid construct as definedaccording to the invention.

In a preferred embodiment of the genetically modified microorganisms,the genes described above are at least one nucleic acid encoding aprotein from the biosynthetic pathway of fine chemicals.

In a particularly preferred embodiment of the genetically modifiedmicroorganisms, the genes described above are selected from the group ofnucleic acids encoding a protein from the biosynthetic pathway ofproteinogenic and non-proteinogenic amino acids, nucleic acids encodinga protein from the biosynthetic pathway of nucleotides and nucleosides,nucleic acids encoding a protein from the biosynthetic pathway oforganic acids, nucleic acids encoding a protein from the biosyntheticpathway of lipids and fatty acids, nucleic acids encoding a protein fromthe biosynthetic pathway of diols, nucleic acids encoding a protein fromthe biosynthetic pathway of carbohydrates, nucleic acids encoding aprotein from the biosynthetic pathway of aromatic compounds, nucleicacids encoding a protein from the biosynthetic pathway of vitamins,nucleic acids encoding a protein from the biosynthetic pathway ofcofactors and nucleic acids encoding a protein from the biosyntheticpathway of enzymes, where the genes may optionally comprise furtherregulatory elements.

Preferred proteins from the biosynthetic pathway of amino acids areselected from the group of aspartate kinase, aspartate-semialdehydedehydrogenase, diaminopimelate dehydrogenase, diaminopimelatedecarboxylase, dihydrodipicolinate synthetase, dihydrodipicolinatereductase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglyceratekinase, pyruvate carboxylase, triosephosphate isomerase, transcriptionalregulator LuxR, transcriptional regulator LysR1, transcriptionalregulator LysR2, malate-quinone oxidoreductase, glucose-6-phosphatedeydrogenase, 6-phosphogluconate dehydrogenase, transketolase,transaldolase, homoserine O-acetyltransferase, cystathioninegamma-synthase, cystathionine beta-lyase, serinehydroxymethyltransferase, O-acetylhomoserine sulfhydrylase,methylenetetrahydrofolate reductase, phosphoserine aminotransferase,phosphoserine phosphatase, serine acetyltransferase, homoserinedehydrogenase, homoserine kinase, threonine synthase, threonine exportercarrier, threonine dehydratase, pyruvate oxidase, lysine exporter,biotin ligase, cysteine synthase 1, cysteine synthase II, coenzymeB12-dependent methionine synthase, coenzyme B12-independent methioninesynthase activity, sulfate adenylyltransferase subunit 1 and 2,phosphoadenosine-phosphosulfate reductase, ferredoxin-sulfite reductase,ferredoxin NADP reductase, 3-phosphoglycerate dehydrogenase, RXA00655regulator, RXN2910 regulator, arginyl-tRNA synthetase,phosphoenolpyruvate carboxylase, threonine efflux protein, serinehydroxymethyltransferase, fructose-1,6-bisphosphatase, protein ofsulfate reduction RXA077, protein of sulfate reduction RXA248, proteinof sulfate reduction RXA247, protein OpcA, 1-phosphofructokinase and6-phosphofructokinase.

Particularly preferred examples of the proteins and genes from thebiosynthetic pathway of amino acids are described above in Table 1 andTable 2.

Preferred microorganisms or genetically modified microorganisms arebacteria, algae, fungi or yeasts.

Particularly preferred microorganisms are, in particular, coryneformbacteria. Preferred coryneform bacteria are bacteria of the genusCorynebacterium, in particular of the species Corynebacteriumglutamicum, Corynebacterium acetoglutamicum, Corynebacteriumacetoacidophilum, Corynebacterium thermoaminogenes, Corynebacteriummelassecola and Corynebacterium efficiens or of the genusBrevibacterium, in particular of the species Brevibacterium flavum,Brevibacterium lactofermentum and Brevibacterium divaricatum.

Particularly preferred bacteria of the genera Corynebacterium andBrevibacterium are selected from the group of Corynebacterium glutamicumATCC 13032, Corynebacterium acetoglutamicum ATCC 15806, Corynebacteriumacetoacidophilum ATCC 13870, Corynebacterium thermoaminogenes FERMBP-1539, Corynebacterium melassecola ATCC 17965, Corynebacteriumefficiens DSM 44547, Corynebacterium efficiens DSM 44548,Corynebacterium efficiens DSM 44549, Brevibacterium flavum ATCC 14067,Brevibacterium lactofermentum ATCC 13869, Brevibacterium divaricatumATCC 14020, Corynebacterium glutamicum KFCC10065 and Corynebacteriumglutamicum ATCC21608.

The abbreviation KFCC means the Korean Federation of Culture Collection,the abbreviation ATCC the American type strain culture collection andthe abbreviation DSM the Deutsche Sammlung von Mikroorganismen.

Further particularly preferred bacteria of the genera Corynebacteriumand Brevibacterium are listed in Table 3:

Bacterium Deposition number Genus species ATCC FERM NRRL CECT NCIMB CBSNCTC DSMZ Brevibacterium ammoniagenes 21054 Brevibacterium ammoniagenes19350 Brevibacterium ammoniagenes 19351 Brevibacterium ammoniagenes19352 Brevibacterium ammoniagenes 19353 Brevibacterium ammoniagenes19354 Brevibacterium ammoniagenes 19355 Brevibacterium ammoniagenes19356 Brevibacterium ammoniagenes 21055 Brevibacterium ammoniagenes21077 Brevibacterium ammoniagenes 21553 Brevibacterium ammoniagenes21580 Brevibacterium ammoniagenes 39101 Brevibacterium butanicum 21196Brevibacterium divaricatum 21792 P928 Brevibacterium flavum 21474Brevibacterium flavum 21129 Brevibacterium flavum 21518 Brevibacteriumflavum B11474 Brevibacterium flavum B11472 Brevibacterium flavum 21127Brevibacterium flavum 21128 Brevibacterium flavum 21427 Brevibacteriumflavum 21475 Brevibacterium flavum 21517 Brevibacterium flavum 21528Brevibacterium flavum 21529 Brevibacterium flavum B11477 Brevibacteriumflavum B11478 Brevibacterium flavum 21127 Brevibacterium flavum B11474Brevibacterium healii 15527 Brevibacterium ketoglutamicum 21004Brevibacterium ketoglutamicum 21089 Brevibacterium ketosoreductum 21914Brevibacterium lactofermentum 70 Brevibacterium lactofermentum 74Brevibacterium lactofermentum 77 Brevibacterium lactofermentum 21798Brevibacterium lactofermentum 21799 Brevibacterium lactofermentum 21800Brevibacterium lactofermentum 21801 Brevibacterium lactofermentum B11470Brevibacterium lactofermentum B11471 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 21420 Brevibacterium lactofermentum 21086Brevibacterium lactofermentum 31269 Brevibacterium linens 9174Brevibacterium linens 19391 Brevibacterium linens 8377 Brevibacteriumparaffinolyticum 11160 Brevibacterium spec. 717.73 Brevibacterium spec.717.73 Brevibacterium spec. 14604 Brevibacterium spec. 21860Brevibacterium spec. 21864 Brevibacterium spec. 21865 Brevibacteriumspec. 21866 Brevibacterium spec. 19240 Corynebacterium acetoacidophilum21476 Corynebacterium acetoacidophilum 13870 Corynebacteriumacetoglutamicum B11473 Corynebacterium acetoglutamicum B11475Corynebacterium acetoglutamicum 15806 Corynebacterium acetoglutamicum21491 Corynebacterium acetoglutamicum 31270 Corynebacterium acetophilumB3671 Corynebacterium ammoniagenes 6872 2399 Corynebacteriumammoniagenes 15511 Corynebacterium fujiokense 21496 Corynebacteriumglutamicum 14067 Corynebacterium glutamicum 39137 Corynebacteriumglutamicum 21254 Corynebacterium glutamicum 21255 Corynebacteriumglutamicum 31830 Corynebacterium glutamicum 13032 Corynebacteriumglutamicum 14305 Corynebacterium glutamicum 15455 Corynebacteriumglutamicum 13058 Corynebacterium glutamicum 13059 Corynebacteriumglutamicum 13060 Corynebacterium glutamicum 21492 Corynebacteriumglutamicum 21513 Corynebacterium glutamicum 21526 Corynebacteriumglutamicum 21543 Corynebacterium glutamicum 13287 Corynebacteriumglutamicum 21851 Corynebacterium glutamicum 21253 Corynebacteriumglutamicum 21514 Corynebacterium glutamicum 21516 Corynebacteriumglutamicum 21299 Corynebacterium glutamicum 21300 Corynebacteriumglutamicum 39684 Corynebacterium glutamicum 21488 Corynebacteriumglutamicum 21649 Corynebacterium glutamicum 21650 Corynebacteriumglutamicum 19223 Corynebacterium glutamicum 13869 Corynebacteriumglutamicum 21157 Corynebacterium glutamicum 21158 Corynebacteriumglutamicum 21159 Corynebacterium glutamicum 21355 Corynebacteriumglutamicum 31808 Corynebacterium glutamicum 21674 Corynebacteriumglutamicum 21562 Corynebacterium glutamicum 21563 Corynebacteriumglutamicum 21564 Corynebacterium glutamicum 21565 Corynebacteriumglutamicum 21566 Corynebacterium glutamicum 21567 Corynebacteriumglutamicum 21568 Corynebacterium glutamicum 21569 Corynebacteriumglutamicum 21570 Corynebacterium glutamicum 21571 Corynebacteriumglutamicum 21572 Corynebacterium glutamicum 21573 Corynebacteriumglutamicum 21579 Corynebacterium glutamicum 19049 Corynebacteriumglutamicum 19050 Corynebacterium glutamicum 19051 Corynebacteriumglutamicum 19052 Corynebacterium glutamicum 19053 Corynebacteriumglutamicum 19054 Corynebacterium glutamicum 19055 Corynebacteriumglutamicum 19056 Corynebacterium glutamicum 19057 Corynebacteriumglutamicum 19058 Corynebacterium glutamicum 19059 Corynebacteriumglutamicum 19060 Corynebacterium glutamicum 19185 Corynebacteriumglutamicum 13286 Corynebacterium glutamicum 21515 Corynebacteriumglutamicum 21527 Corynebacterium glutamicum 21544 Corynebacteriumglutamicum 21492 Corynebacterium glutamicum B8183 Corynebacteriumglutamicum B8182 Corynebacterium glutamicum B12416 Corynebacteriumglutamicum B12417 Corynebacterium glutamicum B12418 Corynebacteriumglutamicum B11476 Corynebacterium glutamicum 21608 Corynebacteriumlilium P973 Corynebacterium nitrilophilus 21419 11594 Corynebacteriumspec. P4445 Corynebacterium spec. P4446 Corynebacterium spec. 31088Corynebacterium spec. 31089 Corynebacterium spec. 31090 Corynebacteriumspec. 31090 Corynebacterium spec. 31090 Corynebacterium spec. 1595420145 Corynebacterium spec. 21857 Corynebacterium spec. 21862Corynebacterium spec. 21863

The abbreviations have the following meaning:

ATCC: American Type Culture Collection, Rockville, Md., USA FERM:Fermentation Research Institute, Chiba, Japan NRRL: ARS CultureCollection, Northern Regional Research Laboratory, Peoria, Ill., USACECT: Coleccion Espanola de Cultivos Tipo, Valencia, Spain NCIMB:National Collection of Industrial and Marine Bacteria Ltd., Aberdeen, UK

CBS: Centraalbureau voor Schimmelcultures, Baarn, NL

NCTC: National Collection of Type Cultures, London, UK DSMZ: DeutscheSammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany

Through the nucleic acids of the invention having promoter activity andthe expression units of the invention it is possible with the aid of themethods of the invention described above to regulate the metabolicpathways in the genetically modified microorganisms of the inventiondescribed above to specific biosynthetic products.

For this purpose, for example, metabolic pathways which lead to aspecific biosynthetic product are enhanced by causing or increasing thetranscription rate or expression rate of genes of this biosyntheticpathway in which the increased quantity of protein leads to an increasedtotal activity of these proteins of the desired biosynthetic pathway andthus to an enhanced metabolic flux toward the desired biosyntheticproduct.

In addition, metabolic pathways which diverge from a specificbiosynthetic product can be diminished by reducing the transcriptionrate or expression rate of genes of this divergent biosynthetic pathwayin which the reduced quantity of protein leads to a reduced totalactivity of these proteins of the unwanted biosynthetic pathway and thusadditionally to an enhanced metabolic flux toward the desiredbiosynthetic product.

The genetically modified microorganisms of the invention are able forexample to produce biosynthetic products from glucose, sucrose, lactose,fructose, maltose, molasses, starch, cellulose or from glycerol andethanol.

The invention therefore relates to a method for producing biosyntheticproducts by cultivating genetically modified microorganisms of theinvention.

Depending on the desired biosynthetic product, the transcription rate orexpression rate of various genes must be increased or reduced.Ordinarily, it is advantageous to alter the transcription rate orexpression rate of a plurality of genes, i.e. to increase thetranscription rate or expression rate of a combination of genes and/orto reduce the transcription rate or expression rate of a combination ofgenes.

In the genetically modified microorganisms of the invention, at leastone altered, i.e. increased or reduced, transcription rate or expressionrate of a gene is attributable to a nucleic acid of the invention havingpromoter activity or expression unit of the invention.

Further, additionally altered, i.e. additionally increased oradditionally reduced, transcription rates or expression rates of furthergenes in the genetically modified microorganism may, but need not,derive from the nucleic acids of the invention having promoter activityor the expression units of the invention.

The invention therefore further relates to a method for producingbiosynthetic products by cultivating genetically modified microorganismsof the invention. Preferred biosynthetic products are fine chemicals.

The term “fine chemical” is known in the art and includes compoundswhich are produced by an organism and are used in various branches ofindustry such as, for example but not restricted to, the pharmaceuticalindustry, the agriculture, cosmetics, food and feed industries. Thesecompounds include organic acids such as, for example, tartaric acid,itaconic acid and diaminopimelic acid, and proteinogenic andnon-proteinogenic amino acids, purine bases and pyrimidine bases,nucleosides and nucleotides (as described for example in Kuninaka, A.(1996) Nucleotides and related compounds, pp. 561-612, in Biotechnologyvol. 6, Rehm et al., editors, VCH: Weinheim and the references presenttherein), lipids, saturated and unsaturated fatty acids (e.g.arachidonic acid), diols (e.g. propanediol and butanediol),carbohydrates (e.g. hyaluronic acid and trehalose), aromatic compounds(e.g. aromatic amines, vanillin and indigo), vitamins and cofactors (asdescribed in Ullmann's Encyclopedia of Industrial Chemistry, vol. A27,“Vitamins”, pp. 443-613 (1996) VCH: Weinheim and the references presenttherein; and Ong, A. S., Niki, E. and Packer, L. (1995) “Nutrition,Lipids, Health and Disease” Proceedings of the UNESCO/Confederation ofScientific and Technological Associations in Malaysia and the Societyfor Free Radical Research—Asia, held on Sep. 1-3, 1994 in Penang,Malaysia, AOCS Press (1995)), enzymes and all other chemicals describedby Gutcho (1983) in Chemicals by Fermentation, Noyes Data Corporation,ISBN: 0818805086 and the references indicated therein. The metabolismand the uses of certain fine chemicals are explained further below.

I. Amino Acid Metabolism and Uses

The amino acids comprise the fundamental structural units of allproteins and are thus essential for normal cell functions. The term“amino acid” is known in the art. The proteinogenic amino acids, ofwhich there are 20 types, serve as structural units for proteins, inwhich they are linked together by peptide bonds, whereas thenon-proteinogenic amino acids (of which hundreds are known) usually donot occur in proteins (see Ullmann's Encyclopedia of IndustrialChemistry, vol. A2, pp. 57-97 VCH: Weinheim (1985)). The amino acids maybe in the D or L configuration, although L-amino acids are usually theonly type found in naturally occurring proteins. Biosynthetic anddegradation pathways of each of the 20 proteinogenic amino acids arewell characterized both in prokaryotic and in eukaryotic cells (see, forexample, Stryer, L. Biochemistry, 3rd edition, pp. 578-590 (1988)). The“essential” amino acids (histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan and valine), so-calledbecause they must, owing to the complexity of their biosynthesis, betaken in with the diet, are converted by simple biosynthetic pathwaysinto the other 11 “nonessential” amino acids (alanine, arginine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine and tyrosine). Higher animals have the ability to synthesize someof these amino acids, but the essential amino acids must be taken inwith the food in order for normal protein synthesis to take place.

Apart from their function in protein biosynthesis, these amino acids arechemicals of interest per se, and it has been found that many have usesin various applications in the food, feed, chemicals, cosmetics,agriculture and pharmaceutical industries. Lysine is an important aminoacid not only for human nutrition but also for monogastric species suchas poultry and pigs. Glutamate is used most frequently as flavoradditive (monosodium glutamate, MSG) and widely in the food industry, aswell as aspartate, phenylalanine, glycine and cysteine. Glycine,L-methionine and tryptophan are all used in the pharmaceutical industry.Glutamine, valine, leucine, isoleucine, histidine, arginine, proline,serine and alanine are used in the pharmaceutical industry and thecosmetics industry. Threonine, tryptophan and D-/L-methionine are widelyused feed additives (Leuchtenberger, W. (1996) Amino acids—technicalproduction and use, pp. 466-502 in Rehm et al., (editors) Biotechnologyvol. 6, chapter 14a, VCH: Weinheim). It has been found that these aminoacids are additionally suitable as precursors for synthesizing syntheticamino acids and proteins such as N-acetylcysteine,S-carboxymethyl-L-cysteine, (S)-5-hydroxytryptophan and other substancesdescribed in Ullmann's Encyclopedia of Industrial Chemistry, vol. A2,pp. 57-97, VCH, Weinheim, 1985.

The biosynthesis of these natural amino acids in organisms able toproduce them, for example bacteria, has been well characterized (for areview of bacterial amino acid biosynthesis and its regulation, seeUmbarger, H. E. (1978) Ann. Rev. Biochem. 47: 533-606). Glutamate issynthesized by reductive amination of α-ketoglutarate, an intermediatein the citric acid cycle. Glutamine, proline and arginine are eachgenerated successively from glutamate. Biosynthesis of serine takesplace in a three-step method and starts with 3-phosphoglycerate (anintermediate of glycolysis) and yields this amino acid after oxidation,transamination and hydrolysis steps. Cysteine and glycine are eachproduced from serine, the former by condensation of homocysteine withserine, and the latter by transfer of the side-chain β-carbon atom totetrahydrofolate in a reaction catalyzed by serinetranshydroxymethylase. Phenylalanine and tyrosine are synthesized fromthe precursors of the glycolysis and pentose phosphate pathways,erythrose 4-phosphate and phosphenolpyruvate in a 9-step biosyntheticpathway which differs only in the last two steps after the synthesis ofprephenate. Tryptophan is likewise produced from these two startingmolecules, but its synthesis takes place in an 11-step pathway. Tyrosinecan also be produced from phenylalanine in a reaction catalyzed byphenylalanine hydroxylase. Alanine, valine and leucine are eachbiosynthetic products of pyruvate, the final product of glycolysis.Aspartate is formed from oxalacetate, an intermediate of the citratecycle. Asparagine, methionine, threonine and lysine are each produced byconversion of aspartate. Isoleucine is formed from threonine. Histidineis formed in a complex 9-step pathway from 5-phosphoribosyl1-pyrophosphate, an activated sugar.

Amino acids whose quantity exceeds the protein biosynthesis requirementof the cell cannot be stored and are instead degraded, so thatintermediates are provided for the main metabolic pathways of the cell(for a review, see Stryer, L., Biochemistry, 3rd edition, chapter 21“Amino Acid Degradation and the Urea Cycle”; pp. 495-516 (1988)).Although the cell is able to convert unwanted amino acids into usefulmetabolic intermediates, amino acid production is costly in terms of theenergy, the precursor molecules and the enzymes required for theirsynthesis. It is therefore not surprising that amino acid biosynthesisis regulated by feedback inhibition, where the presence of a particularamino acid slows down or entirely terminates its own production (for areview of the feedback mechanism in amino acid biosynthetic pathways,see Stryer, L., Biochemistry, 3rd edition, chapter 24, “Biosynthesis ofAmino Acids and Heme”, pp. 575-600 (1988)). The output of a particularamino acid is therefore restricted by the quantity of this amino acid inthe cell.

II. Vitamins, Cofactors and Nutraceutical Metabolism, and Uses

Vitamins, cofactors and nutraceuticals comprise a further group ofmolecules. Higher animals have lost the ability to synthesize these andtherefore need to take them in, although they are easily synthesized byother organisms such as bacteria. These molecules are eitherbiologically active molecules per se or precursors of biologicallyactive substances which serve as electron carriers or intermediates in anumber of metabolic pathways. These compounds have, besides theirnutritional value, also a significant industrial value as coloringagents, antioxidants and catalysts or other processing aids. (For areview of the structure, activity and industrial applications of thesecompounds, see, for example, Ullmann's Encyclopedia of IndustrialChemistry, “Vitamins”, vol. A27, pp. 443-613, VCH: Weinheim, 1996). Theterm “vitamin” is known in the art and includes nutrients which arerequired by an organism for normal functioning, but cannot besynthesized by this organism itself. The group of vitamins may includecofactors and nutraceutical compounds. The term “cofactor” includesnon-protein compounds which are necessary for the occurrence of normalenzymic activity. These compounds may be organic or inorganic; thecofactor molecules of the invention are preferably organic. The term“nutraceutical” includes food additives which promote health in plantsand animals, especially in humans. Examples of such molecules arevitamins, antioxidants and likewise certain lipids (e.g. polyunsaturatedfatty acids).

Biosynthesis of these molecules in organisms able to produce them, suchas bacteria, has been characterized in detail (Ullmann's Encyclopedia ofIndustrial Chemistry, “Vitamins”, vol. A27, pp. 443-613, VCH: Weinheim,1996, Michal, G. (1999) Biochemical Pathways An Atlas of Biochemistryand Molecular Biology, John Wiley & Sons; Ong, A. S., Niki, E. andPacker, L. (1995) “Nutrition, Lipids, Health and Disease” Proceedings ofthe UNESCO/Confederation of Scientific and Technological Associations inMalaysia and the Society for free Radical Research—Asia, held on Sep.1-3, 1994, in Penang, Malaysia, AOCS Press, Champaign, Ill. X, 374 S).

Thiamine (vitamin B₁) is formed by chemical coupling of pyrimidine andthiazole units. Riboflavin (vitamin B₂) is synthesized from guanosine5′-triphosphate (GTP) and ribose 5-phosphate. Riboflavin in turn isemployed for the synthesis of flavin mononucleotide (FMN) andflavin-adenine dinucleotide (FAD). The family of compounds referred tojointly as “vitamin B6” (e.g. pyridoxine, pyridoxamine, pyridoxal5-phosphate and the commercially used pyridoxine hydrochloride) are allderivatives of the common structural unit 5-hydroxy-6-methylpyridine.Pantothenate (pantothenic acid,R-(+)-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-β-alanine) can beproduced either by chemical synthesis or by fermentation. The last stepsin pantothenate biosynthesis consist of ATP-driven condensation ofβ-alanine and pantoic acid. The enzymes responsible for the biosyntheticsteps for conversion into pantoic acid, into β-alanine and forcondensation to pantothenic acid are known. The metabolically activeform of pantothenate is coenzyme A, whose biosynthesis proceeds through5 enzymatic steps. Pantothenate, pyridoxal 5-phosphate, cysteine and ATPare the precursors of coenzyme A. These enzymes catalyze not only theformation of pantothenate but also the production of (R)-pantoic acid,(R)-pantolactone, (R)-panthenol (provitamin B₅), pantethein (and itsderivatives) and coenzyme A.

The biosynthesis of biotin from the precursor molecule pimeloyl-CoA inmicroorganisms has been investigated in detail, and several of the genesinvolved have been identified. It has emerged that many of thecorresponding proteins are involved in Fe cluster synthesis and belongto the class of nifS proteins. Lipoic acid is derived from octanoic acidand serves as coenzyme in energy metabolism, where it becomes aconstituent of the pyruvate dehydrogenase complex and of theα-ketoglutarate dehydrogenase complex. The folates are a group ofsubstances which are all derived from folic acid, which in turn isderived from L-glutamic acid, p-aminobenzoic acid and 6-methylpterin.The biosynthesis of folic acid and its derivatives starting from themetabolic intermediates guanosine 5′-triphosphate (GTP), L-glutamic acidand p-aminobenzoic acid has been investigated in detail in certainmicroorganisms.

Corrinoids (such as the cobalamins and in particular vitamin B₁₂) andthe porphyrins belong to a group of chemicals which are distinguished bya tetrapyrrole ring system. The biosynthesis of vitamin B₁₂ is socomplex that it has not yet been completely characterized, but most ofthe enzymes and substrates involved are now known. Nicotinic acid(nicotinate) and nicotinamide are pyridine derivatives, which are alsoreferred to as “niacin”. Niacin is the precursor of the importantcoenzymes NAD (nicotinamide-adenine dinucleotide) and NADP(nicotinamide-adenine dinucleotide phosphate) and their reduced forms.

The production of these compounds on the industrial scale is based forthe most part on cell-free chemical syntheses, although some of thesechemicals have likewise been produced by large-scale culturing ofmicroorganisms, such as riboflavin, vitamin B₆, pantothenate and biotin.Only vitamin B₁₂ is produced solely by fermentation, because of thecomplexity of its synthesis. In vitro methods require a considerableexpenditure of materials and time and frequently of high costs.

III. Purine, Pyrimidine, Nucleoside and Nucleotide Metabolism and Uses

Genes for purine and pyrimidine metabolism and their correspondingproteins are important targets for the therapy of neoplastic diseasesand viral infections. The term “purine” or “pyrimidine” comprisesnitrogenous bases which are a constituent of nucleic acids, coenzymesand nucleotides. The term “nucleotide” comprises the fundamentalstructural units of nucleic acid molecules, which include a nitrogenousbase, a pentose sugar (the sugar in RNA is ribose, and the sugar in DNAis D-deoxyribose) and phosphoric acid. The term “nucleoside” comprisesmolecules which serve as precursors of nucleotides but which, incontrast to nucleotides, have no phosphoric acid unit. It is possible byinhibiting the biosynthesis of these molecules or their mobilization forformation of nucleic acid molecules to inhibit RNA and DNA synthesis;targeted inhibition of this activity in carcinogenic cells allows theability of tumor cells to divide and replicate to be inhibited.

There are also nucleotides which do not form nucleic acid molecules butserve as energy stores (i.e. AMP) or as coenzymes (i.e. FAD and NAD).

Several publications have described the use of these chemicals for thesemedical indications, where purine and/or pyrimidine metabolism isinfluenced (e.g. Christopherson, R. I. and Lyons, S.D. (1990) “Potentinhibitors of de novo pyrimidine and purine biosynthesis aschemotherapeutic agents”, Med. Res. Reviews 10: 505-548). Investigationson enzymes involved in purine and pyrimidine metabolism haveconcentrated on the development of novel medicaments which can be usedfor example as immunosuppressants or antiproliferatives (Smith, J. L.“Enzymes in Nucleotide Synthesis” Curr. Opin. Struct. Biol. 5 (1995)752-757; Biochem. Soc. Transact. 23 (1995) 877-902). Purine andpyrimidine bases, nucleosides and nucleotides have, however, also otherpossible uses: as intermediates in the biosynthesis of various finechemicals (e.g. thiamine, S-adenosylmethionine, folates or riboflavin),as energy carriers for the cell (e.g. ATP or GTP) and for chemicalsthemselves, are commonly used as flavor enhancers (e.g. IMP or GMP) orfor many medical applicatons (see, for example, Kuninaka, A., (1996)“Nucleotides and Related Compounds” in Biotechnology, vol. 6, Rehm etal., editors VCH: Weinheim, pp. 561-612). Enzymes involved in purine,pyridine, nucleoside or nucleotide metabolism are also increasinglyserving as targets for the development of chemicals for crop protection,including fungicides, herbicides and insecticides.

The metabolism of these compounds in bacteria has been characterized(for reviews, see, for example, Zalkin, H. and Dixon, J. E. (1992) “Denovo purine nucleotide biosynthesis” in Progress in Nucleic AcidsResearch and Molecular biology, vol. 42, Academic Press, pp. 259-287;and Michal, G. (1999) “Nucleotides and Nucleosides”; chapter 8 in:Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology,Wiley, New York). Purine metabolism, which is the subject of intensiveresearch, is essential for normal functioning of the cell. Impairedpurine metabolism in higher animals may cause severe disorders, e.g.gout. The purine nucleotides are synthesized over a number of steps viathe intermediate compound inosine 5′-phosphate (IMP) from ribose5-phosphate, leading to production of guanosine 5′-monophosphate (GMP)or adenosine 5′-monophosphate (AMP), from which the triphosphate forms,which are used as nucleotides, can easily be prepared. These compoundsare also used as energy stores, so that their degradation providesenergy for many different biochemical processes in the cell. Pyrimidinebiosynthesis takes place via the formation of uridine 5′-monophosphate(UMP) from ribose 5-phosphate. UMP in turn is converted into cytidine5′-triphosphate (CTP). The deoxy forms of all nucleotides are preparedin a one-step reduction reaction from the diphosphate ribose form of thenucleotide to give the diphosphate deoxyribose form of the nucleotide.After phosphorylation, these molecules are able to take part in DNAsynthesis.

IV. Trehalose Metabolism and Uses

Trehalose consists of two glucose molecules which are linked togethervia an α,α-1,1 linkage. It is commonly used in the food industry assweetener, as additive to dried or frozen foods and in beverages.However, it is also used in the pharmaceutical industry, the cosmeticsand biotechnology industry (see, for example, Nishimoto et al., (1998)U.S. Pat. No. 5,759,610; Singer, M. A. and Lindquist, S. Trends Biotech.16 (1998) 460-467; Paiva, C. L. A. and Panek, A. D. Biotech Ann. Rev. 2(1996) 293-314; and Shiosaka, M. FFIJ. Japan 172 (1997) 97-102).Trehalose is produced by enzymes of many microorganisms and is releasedin a natural way into the surrounding medium, from which it can beisolated by methods known in the art.

Particularly preferred biosynthetic products are selected from the groupof organic acids, proteins, nucleotides and nucleosides, bothproteinogenic and non-proteinogenic amino acids, lipids and fatty acids,diols, carbohydrates, aromatic compounds, vitamins and cofactors,enzymes and proteins.

Preferred organic acids are tartaric acid, itaconic acid anddiaminopimelic acid.

Preferred nucleosides and nucleotides are described for example inKuninaka, A. (1996) Nucleotides and related compounds, pp. 561-612, inBiotechnology, vol. 6, Rehm et al., editors VCH: Weinheim and referencespresent therein.

Preferred biosynthetic products are additionally lipids, saturated andunsaturated fatty acids such as, for example, arachidonic acid, diolssuch as, for example, propanediol and butanediol, carbohydrates such as,for example, hyaluronic acid and trehalose, aromatic compounds such as,for example, aromatic amines, vanillin and indigo, vitamins andcofactors as described for example in Ullmann's Encyclopedia ofIndustrial Chemistry, vol. A27, “Vitamins”, pp. 443-613 (1996) VCH:Weinheim and the references present therein; and Ong, A. S., Niki, E.and Packer, L. (1995) “Nutrition, Lipids, Health and Disease”Proceedings of the UNESCO/Confederation of Scientific and TechnologicalAssociations in Malaysia and the Society for Free Radical Research—Asia,held on Sep. 1-3, 1994 in Penang, Malaysia, AOCS Press (1995)), enzymes,polyketides (Cane et al. (1998) Science 282: 63-68) and all otherchemicals described by Gutcho (1983) in Chemicals by Fermentation, NoyesData Corporation, ISBN: 0818805086 and the references indicated therein.

Particularly preferred biosynthetic products are amino acids,particularly preferably essential amino acids, in particular L-glycine,L-alanine, L-leucine, L-methionine, L-phenylalanine, L-tryptophan,L-lysine, L-glutamine, L-glutamic acid, L-serine, L-proline, L-valine,L-isoleucine, L-cysteine, L-tyrosine, L-histidine, L-arginine,L-asparagine, L-aspartic acid and L-threonine, L-homoserine, especiallyL-lysine, L-methionine and L-threonine. An amino acid such as, forexample, lysine, methionine and threonine means hereinafter both in eachcase the L and the D form of the amino acid, preferably the L form, i.e.for example L-lysine, L-methionine and L-threonine.

The invention relates in particular to a method for producing lysine bycultivating genetically modified microorganisms with increased or causedexpression rate of at least one gene compared with the wild type, where

ch) the specific expression activity in the microorganism of at leastone endogenous expression unit of the invention, which regulates theexpression of the endogenous genes, is increased compared with the wildtype, ordh) the expression of genes in the microorganism is regulated byexpression units of the invention or by expression units with increasedspecific expression activity as set forth in embodiment a), where thegenes are heterologous in relation to the expression units,and where the genes are selected from the group of nucleic acidsencoding an aspartate kinase, nucleic acids encoding anaspartate-semialdehyde dehydrogenase, nucleic acids encoding adiaminopimelate dehydrogenase, nucleic acids encoding a diaminopimelatedecarboxylase, nucleic acids encoding a dihydrodipicolinate synthetase,nucleic acids encoding a dihydrodipicolinate reductase, nucleic acidsencoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acidsencoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvatecarboxylase, nucleic acids encoding a triosephosphate isomerase, nucleicacids encoding a transcriptional regulator LuxR, nucleic acids encodinga transcriptional regulator LysR1, nucleic acids encoding atranscriptional regulator LysR2, nucleic acids encoding a malate-quinoneoxidoreductase, nucleic acids encoding a glucose-6-phosphatedehydrogenase, nucleic acids encoding a 6-phosphogluconatedehydrogenase, nucleic acids encoding a transketolase, nucleic acidsencoding a transaldolase, nucleic acids encoding a lysine exporter,nucleic acids encoding a biotin ligase, nucleic acids encoding anarginyl-tRNA synthetase, nucleic acids encoding a phosphoenolpyruvatecarboxylase, nucleic acids encoding a fructose-1,6-bisphosphatase,nucleic acids encoding a protein OpcA, nucleic acids encoding a1-phosphofructokinase and nucleic acids encoding a6-phosphofructokinase.

As described above for the methods, the regulation of the expression ofthese genes in the microorganism by expression units of the invention orby expression units of the invention with increased specific expressionactivity in accordance with embodiment ch) is achieved by

dh1) introducing one or more expression units of the invention, whereappropriate with increased specific expression activity, into the genomeof the microorganism so that expression of one or more endogenous genestakes place under the control of the introduced expression units of theinvention, where appropriate with increased specific expressionactivity, ordh2) introducing one or more of these genes into the genome of themicroorganism so that expression of one or more of the introduced genestakes place under the control of the endogenous expression units of theinvention, where appropriate with increased specific expressionactivity, ordh3) introducing one or more nucleic acid constructs comprising anexpression unit of the invention, where appropriate with increasedspecific expression activity, and functionally linked one or morenucleic acids to be expressed, into the microorganism.

A further preferred embodiment of the method described above forpreparing lysine comprises the genetically modified microorganisms,compared with the wild type, having additionally an increased activity,of at least one of the activities selected from the group of aspartatekinase activity, aspartate-semialdehyde dehydrogenase activity,diaminopimelate dehydrogenase activity, diaminopimelate decarboxylaseactivity, dihydrodipicolinate synthetase activity, dihydrodipicolinatereductase activity, glyceraldehyde-3-phosphate dehydrogenase activity,3-phosphoglycerate kinase activity, pyruvate carboxylase activity,triosephosphate isomerase activity, activity of the transcriptionalregulator LuxR, activity of the transcriptional regulator LysR1,activity of the transcriptional regulator LysR2, malate-quinoneoxidoreductase activity, glucose-6-phosphate dehydrogenase activity,6-phosphogluconate dehydrogenase activity, transketolase activity,transaldolase activity, lysine exporter activity, arginyl-tRNAsynthetase activity, phosphoenolpyruvate carboxylase activity,fructose-1,6-bisphosphatase activity, protein OpcA activity,1-phosphofructokinase activity, 6-phosphofructokinase activity andbiotin ligase activity.

A further particularly preferred embodiment of the method describedabove for preparing lysine comprises the genetically modifiedmicroorganisms having, compared with the wild type, additionally areduced activity, of at least one of the activities selected from thegroup of threonine dehydratase activity, homoserine O-acetyl-transferaseactivity, O-acetylhomoserine sulfhydrylase activity, phosphoenolpyruvatecarboxykinase activity, pyruvate oxidase activity, homoserine kinaseactivity, homoserine dehydrogenase activity, threonine exporteractivity, threonine efflux protein activity, asparaginase activity,aspartate decarboxylase activity and threonine synthase activity.

These additional increased or reduced activities of at least one of theactivities described above may, but need not, be caused by a nucleicacid of the invention having promoter activity and/or an expression unitof the invention.

The invention further relates to a method for producing methionine bycultivating genetically modified microorganisms with increased or causedexpression rate of at least one gene compared with the wild type, where

ch) the specific expression activity in the microorganism of at leastone endogenous expression unit of the invention, which regulates theexpression of the endogenous genes, is increased compared with the wildtype, ordh) the expression of genes in the microorganism is regulated byexpression units of the invention or by expression units of theinvention with increased specific expression activity as set forth inembodiment a), where the genes are heterologous in relation to theexpression units,and where the genes are selected from the group of nucleic acids encodngan aspartate kinase, nucleic acids encoding an aspartate-semialdehydedehydrogenase, nucleic acids encoding a homoserine dehydrogenase,nucleic acids encoding a glyceraldehyde-3-phosphate dehydrogenase,nucleic acids encoding a 3-phosphoglycerate kinase, nucleic acidsencoding a pyruvate carboxylase, nucleic acids encoding atriosephosphate isomerase, nucleic acids encoding a homoserineO-acetyltransferase, nucleic acids encoding a cystathioninegamma-synthase, nucleic acids encoding a cystathionine beta-lyase,nucleic acids encoding a serine hydroxymethyltransferase, nucleic acidsencoding an O-acetylhomoserine sulfhydrylase, nucleic acids encoding amethylenetetrahydrofolate reductase, nucleic acids encoding aphosphoserine aminotransferase, nucleic acids encoding a phosphoserinephosphatase, nucleic acids encoding a serine acetyltransferase, nucleicacids encoding a cysteine synthase 1, nucleic acids encoding a cysteinesynthase II activity, nucleic acids encoding a coenzyme B12-dependentmethionine synthase activity, nucleic acids encoding a coenzymeB12-independent methionine synthase activity, nucleic acids encoding asulfate adenylyltransferase activity, nucleic acids encoding aphosphoadenosine phosphosulfate reductase activity, nucleic acidsencoding a ferredoxin-sulfite reductase activity, nucleic acids encodinga ferredoxin NADPH-reductase activity, nucleic acids encoding aferredoxin activity, nucleic acids encoding a protein of sulfatereduction RXA077, nucleic acids encoding a protein of sulfate reductionRXA248, nucleic acids encoding a protein of sulfate reduction RXA247,nucleic acids encoding an RXA0655 regulator and nucleic acids encodingan RXN2910 regulator.

As described above for the methods, the regulation of the expression ofthese genes in the microorganism by expression units of the invention orby expression units of the invention with increased specific expressionactivity according to embodiment ch) is achieved by

dh1) introducing one or more expression units of the invention, whereappropriate with increased specific expression activity, into the genomeof the microorganism so that expression of one or more of theseendogenous genes takes place under the control of the introducedexpression units of the invention, where appropriate with increasedspecific expression activity, ordh2) introducing one or more genes into the genome of the microorganismso that expression of one or more of the introduced genes takes placeunder the control of the endogenous expression units of the invention,where appropriate with increased specific expression activity, ordh3) introducing one or more nucleic acid constructs comprising anexpression unit of the invention, where appropriate with increasedspecific expression activity, and functionally linked one or morenucleic acids to be expressed, into the microorganism.

A further preferred embodiment of the method described above forpreparing methionine comprises the genetically modified microorganismshaving, compared with the wild type, additionally an increased activity,of at least one of the activities selected from the group of aspartatekinase activity, aspartate-semialdehyde dehydrogenase activity,homoserine dehydrogenase activity, glyceraldehyde-3-phosphatedehydrogenase activity, 3-phosphoglycerate kinase activity; pyruvatecarboxylase activity, triosephosphate isomerase activity, homoserineO-acetyltransferase activity, cystathionine gamma-synthase activity,cystathionine beta-lyase activity, serine hydroxymethyltransferaseactivity, O-acetylhomoserine sulfhydrylase activity,methylenetetrahydrofolate reductase activity, phosphoserineaminotransferase activity, phosphoserine phosphatase activity, serineacetyltransferase activity, cysteine synthase I activity, cysteinesynthase II activity, coenzyme B12-dependent methionine synthaseactivity, coenzyme B12-independent methionine synthase activity, sulfateadenylyltransferase activity, phosphoadenosine-phosphosulfate reductaseactivity, ferredoxin-sulfite reductase activity, ferredoxinNADPH-reductase activity, ferredoxin activity, activity of a protein ofsulfate reduction RXA077, activity of a protein of sulfate reductionRXA248, activity of a protein of sulfate reduction RXA247, activity ofan RXA655 regulator and activity of an RXN2910 regulator.

A further particularly preferred embodiment of the method describedabove for preparing methionine comprises the genetically modifiedmicroorganisms having, compared with the wild type, additionally areduced activity, of at least one of the activities selected from thegroup of homoserine kinase activity, threonine dehydratase activity,threonine synthase activity, meso-diaminopimelate D-dehydrogenaseactivity, phosphoenolpyruvate carboxykinase activity, pyruvate oxidaseactivity, dihydrodipicolinate synthase activity, dihydrodipicolinatereductase activity and diaminopicolinate decarboxylase activity.

These additional increased or reduced activities of at least one of theactivities described above may, but need not, be caused by a nucleicacid of the invention having promoter activity and/or an expression unitof the invention.

The invention further relates to a method for preparing threonine bycultivating genetically modified microorganisms with increased or causedexpression rate of at least one gene compared with the wild type, where

ch) the specific expression activity in the microorganism of at leastone endogenous expression unit of the invention, which regulates theexpression of the endogenous genes, is increased compared with the wildtype, ordh) the expression of genes in the microorganism is regulated byexpression units of the invention or by expression units of theinvention with increased specific expression activity as set forth inembodiment a), where the genes are heterologous in relation to theexpression units,and where the genes are selected from the group of nucleic acidsencoding an aspartate kinase, nucleic acids encoding anaspartate-semialdehyde dehydrogenase, nucleic acids encoding aglyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a3-phosphoglycerate kinase, nucleic acids encoding a pyruvatecarboxylase, nucleic acids encoding a triosephosphate isomerase, nucleicacids encoding a homoserine kinase, nucleic acids encoding a threoninesynthase, nucleic acids encoding a threonine exporter carrier, nucleicacids encoding a glucose-6-phosphate dehydrogenase, nucleic acidsencoding a transaldolase, nucleic acids encoding a transketolase,nucleic acids encoding a malate-quinone oxidoreductase, nucleic acidsencoding a 6-phosphogluconate dehydrogenase, nucleic acids encoding alysine exporter, nucleic acids encoding a biotin ligase, nucleic acidsencoding a phosphoenolpyruvate carboxylase, nucleic acids encoding athreonine efflux protein, nucleic acids encoding afructose-1,6-bisphosphatase, nucleic acids encoding an OpcA protein,nucleic acids encoding a 1-phosphofructokinase, nucleic acids encoding a6-phosphofructokinase, and nucleic acids encoding a homoserinedehydrogenase.

As described above for the methods, the regulation of the expression ofthese genes in the microorganism by expression units of the invention orby expression units of the invention with increased specific expressionactivity according to embodiment ch) is achieved by

dh1) introducing one or more expression units of the invention, whereappropriate with increased specific expression activity, into the genomeof the microorganism so that expression of one or more of theseendogenous genes takes place under the control of the introducedexpression units of the invention, where appropriate with increasedspecific expression activity, ordh2) introducing one or more of these genes into the genome of themicroorganism so that expression of one or more of the introduced genestakes place under the control of the endogenous expression units of theinvention, where appropriate with increased specific expressionactivity, ordh3) introducing one or more nucleic acid constructs comprising anexpression unit of the invention, where appropriate with increasedspecific expression activity, and functionally linked one or morenucleic acids to be expressed, into the microorganism.

A further preferred embodiment of the method described above forpreparing threonine comprises the genetically modified microorganismshaving, compared with the wild type, additionally an increased activity,of at least one of the activities selected from the group of aspartatekinase activity, aspartate-semialdehyde dehydrogenase activity,glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglyceratekinase activity, pyruvate carboxylase activity, triosephosphateisomerase activity, threonine synthase activity, activity of a threonineexport carrier, transaldolase activity, transketolase activity,glucose-6-phosphate dehydrogenase activity, malate-quinoneoxidoreductase activity, homoserine kinase activity, biotin ligaseactivity, phosphoenolpyruvate carboxylase activity, threonine effluxprotein activity, protein OpcA activity, 1-phosphofructokinase activity,6-phosphofructokinase activity, fructose-1,6-bisphosphatase activity,6-phosphogluconate dehydrogenase and homoserine dehydrogenase activity.

A further particularly preferred embodiment of the method describedabove for preparing threonine comprises the genetically modifiedmicroorganisms having, compared with the wild type, additionally areduced activity, of at least one of the activities selected from thegroup of threonine dehydratase activity, homoserine O-acetyltransferaseactivity, serine hydroxymethyltransferase activity, O-acetylhomoserinesulfhydrylase activity, meso-diaminopimelate D-dehydrogenase activity,phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity,dihydrodipicolinate synthetase activity, dihydrodipicolinate reductaseactivity, asparaginase activity, aspartate decarboxylase activity,lysine exporter activity, acetolactate synthase activity, ketol-acidreductoisomerase activity, branched chain aminotransferase activity,coenzyme B12-dependent methionine synthase activity, coenzymeB12-independent methionine synthase activity, dihydroxy-acid dehydrataseactivity and diaminopicolinate decarboxylase activity.

These additional increased or reduced activities of at least one of theactivities described above may, but need not, be caused by a nucleicacid of the invention having promoter activity and/or an expression unitof the invention.

The term “activity” of a protein means in the case of enzymes theenzymic activity of the corresponding protein, and in the case of otherproteins, for example structural or transport proteins, thephysiological activity of the proteins.

The enzymes are ordinarily able to convert a substrate into a product orcatalyze this conversion step.

Accordingly, the “activity” of an enzyme means the quantity of substrateconverted by the enzyme, or the quantity of product formed, in aparticular time.

Thus, where an activity is increased compared with the wild type, thequantity of the substrate converted by the enzyme, or the quantity ofproduct formed, in a particular time is increased compared with the wildtype.

This increase in the “activity” preferably amounts, for all activitiesdescribed hereinbefore and hereinafter, to at least 5%, furtherpreferably at least 20%, further preferably at least 50%, furtherpreferably at least 100%, more preferably at least 300%, even morepreferably at least 500%, especially at least 600% of the “activity ofthe wild type”.

Thus, where an activity is reduced compared with the wild type, thequantity of substrate converted by the enzyme, or the quantity ofproduct formed, in a particular time is reduced compared with the wildtype.

A reduced activity preferably means the partial or substantiallycomplete suppression or blocking, based on various cell biologicalmechanisms, of the functionality of this enzyme in a microorganism.

A reduction in the activity comprises a quantitative decrease in anenzyme as far as substantially complete absence of the enzyme (i.e. lackof detectability of the corresponding activity or lack of immunologicaldetectability of the enzyme). The activity in the microorganism ispreferably reduced, compared with the wild type, by at least 5%, furtherpreferably by at least 20%, further preferably by at least 50%, furtherpreferably by 100%. “Reduction” also means in particular the completeabsence of the corresponding activity.

The activity of particular enzymes in genetically modifiedmicroorganisms and in the wild type, and thus the increase or reductionin the enzymic activity, can be measured by known methods such as, forexample, enzyme assays.

For example, a pyruvate carboxylase means a protein which exhibits theenzymatic activity of converting pyruvate into oxaloacetate.

Correspondingly, a pyruvate carboxylase activity means the quantity ofpyruvate converted by the pyruvate carboxlyase protein, or quantity ofoxaloacetate formed, in a particular time.

Thus, where a pyruvate carboxylase activity is increased compared withthe wild type, the quantity of pyruvate converted by the pyruvatecarboxylase protein, or the quantity of oxaloacetate formed, in aparticular time is increased compared with the wild type. This increasein the pyruvate carboxylase activity is preferably at least 5%, furtherpreferably at least 20%, further preferably at least 50%, furtherpreferably at least 100%, more preferably at least 300%, even morepreferably at least 500%, in particular at least 600%, of the pyruvatecarboxylase activity of the wild type.

In addition, for example a phosphoenolpyruvate carboxykinase activitymeans the enzymic activity of a phosphoenolpyruvate carboxykinase.

A phosphoenolpyruvate carboxykinase means a protein which exhibits theenzymatic activity of converting oxaloacetate into phosphoenolpyruvate.

Correspondingly, phosphoenolpyruvate carboxykinase activity means thequantity of oxaloacetate converted by the phosphoenolpyruvatecarboxykinase protein, or quantity of phosphoenolpyruvate formed, in aparticular time.

When the phosphoenolpyruvate carboxykinase activity is reduced comparedwith the wild type, therefore, the quantity of oxaloacetate converted bythe phosphoenolpyruvate carboxykinase protein, or the quantity ofphosphoenolpyruvate formed, in a particular time, is reduced comparedwith the wild type.

A reduction in phosphoenolpyruvate carboxykinase activity comprises aquantitative decrease in a phosphoenolpyruvate carboxykinase as far as asubstantially complete absence of phosphoenolpyruvate carboxykinase(i.e. lack of detectability of phosphoenolpyruvate carboxykinaseactivity or lack of immunological detectability of phosphoenolpyruvatecarboxykinase). The phosphoenolpyruvate carboxykinase activity ispreferably reduced, compared with the wild type, by at least 5%, furtherpreferably by at least 20%, further preferably by at least 50%, furtherpreferably by 100%. In particular, “reduction” also means the completeabsence of phosphoenolpyruvate carboxykinase activity.

The additional increasing of activities can take place in various ways,for example by switching off inhibitory regulatory mechanisms at theexpression and protein level or by increasing gene expression of nucleicacids encoding the proteins described above compared with the wild type.

Increasing the gene expression of the nucleic acids encoding theproteins described above compared with the wild type can likewise takeplace in various ways, for example by inducing the gene by activatorsor, as described above, by increasing the promoter activity orincreasing the expression activity or by introducing one or more genecopies into the microorganism.

Increasing the gene expression of a nucleic acid encoding a protein alsomeans according to the invention manipulation of the expression ofendogenous proteins intrinsic to the microorganism.

This can be achieved for example, as described above, by altering thepromoter and/or expression unit sequences of the genes. Such analteration, which results in an increased expression rate of the gene,can take place for example by deletion or insertion of DNA sequences.

It is possible, as described above, to alter the expression ofendogenous proteins by applying exogenous stimuli. This can take placethrough particular physiological conditions, i.e. through theapplication of foreign substances.

The skilled worker may have recourse to further different procedures,singly or in combination, to achieve an increase in gene expression.Thus, for example, the copy number of the appropriate genes can beincreased, or the promoter and regulatory region or the ribosome bindingsite located upstream of the structural gene can be mutated. It isadditionally possible to increase the expression during fermentativeproduction through inducible promoters. Procedures to prolong thelifespan of the mRNA likewise improve expression. Enzymic activity islikewise enhanced also by preventing degradation of enzyme protein. Thegenes or gene constructs may be either present in plasmids with varyingcopy number or integrated and amplified in the chromosome. It is alsopossible as an alternative to achieve overexpression of the relevantgenes by altering the composition of the media and management of theculture.

The skilled worker can find guidance on this inter alia in Martin et al.(Biotechnology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41(1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), inEikmanns et al. (Gene 102, 93-98 (1991)), in European patent 0472869, inU.S. Pat. No. 4,601,893, in Schwarzer and Pühler (Biotechnology 9, 84-87(1991), in Reinscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994), in LaBarre et al. (Journal of Bacteriology 175,1001-1007 (1993)), in the patent application WO 96/15246, in Malumbreset al. (Gene 134, 15-24 (1993)), in the Japanese published specificationJP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering58, 191-195 (1998)), in Makrides (Microbiological Reviews 60: 512-538(1996) and in well-known textbooks of genetics and molecular biology.

It may additionally be advantageous for the production of biosyntheticproducts, especially L-lysine, L-methionine and L-threonine, besides theexpression or enhancement of a gene, to eliminate unwanted sidereactions (Nakayama: “Breeding of Amino Acid Producing Microorganisms”,in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek(eds.), Academic Press, London, UK, 1982).

In a preferred embodiment, gene expression of a nucleic acid encodingone of the proteins described above is increased by introducing at leastone nucleic acid encoding a corresponding protein into themicroorganism. The introduction of the nucleic acid can take placechromosomally or extrachromosomally, i.e. through increasing the copynumber on the chromosome and/or a copy of the gene on a plasmid whichreplicates in the host microorganism.

The introduction of the nucleic acid, for example in the form of anexpression cassette comprising the nucleic acid, preferably takes placechromosomally, in particular by the SacB method described above.

It is possible in principle to use for this purpose any gene whichencodes one of the proteins described above.

In the case of genomic nucleic acid sequences from eukaryotic sourceswhich comprise introns, if the host microorganism is unable or cannot bemade able to express the corresponding proteins it is preferred to usenucleic acid sequences which have already been processed, such as thecorresponding cDNAs.

Examples of the corresponding genes are listed in Table 1 and 2.

The activities described above in microorganisms are preferably reducedby at least one of the following methods:

-   -   introduction of at least one sense ribonucleic acid sequence for        inducing cosuppression or of an expression cassette ensuring        expression thereof    -   introduction of at least one DNA- or protein-binding factor        against the corresponding gene, RNA or protein or of an        expression cassette ensuring expression thereof    -   introduction of at least one viral nucleic acid sequence which        causes RNA degradation, or of an expression cassette ensuring        expression thereof    -   introduction of at least one construct to produce a loss of        function, such as, for example, generation of stop codons or a        shift in the reading frame, of a gene, for example by producing        an insertion, deletion, inversion or mutation in a gene. It is        possible and preferred to generate knockout mutants by targeted        insertion into the desired target gene through homologous        recombination or introduction of sequence-specific nucleases        against the target gene.    -   introduction of a promoter with reduced promoter activity or of        an expression unit with reduced expression activity.

The skilled worker is aware that further methods can also be employedwithin the scope of the present invention for reducing its activity orfunction. For example, the introduction of a dominant negative variantof a protein or of an expression cassette ensuring expression thereofmay also be advantageous.

It is moreover possible for each single one of these methods to bringabout a reduction in the quantity of protein, quantity of mRNA and/oractivity of a protein. A combined use is also conceivable. Furthermethods are known to the skilled worker and may comprise impeding orsuppressing the processing of the protein, of the transport of theprotein or its mRNA, inhibition of ribosome attachment, inhibition ofRNA splicing, induction of an RNA-degrading enzyme and/or inhibition oftranslation elongation or termination.

In the method of the invention for producing biosynthetic products, thestep of cultivation of the genetically modified microorganisms ispreferably followed by an isolation of biosynthetic products from themicroorganisms or/or from the fermentation broth. These steps may takeplace at the same time and/or preferably after the cultivation step.

The genetically modified microorganisms of the invention can becultivated to produce biosynthetic products, in particular L-lysine,L-methionine and L-threonine, continuously or discontinuously in a batchmethod (batch cultivation) or in the fed batch or repeated fed batchmethod. A summary of known cultivation methods is to be found in thetextbook by Chmiel (Bioprozeβechnik 1. Einführung in dieBioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in thetextbook by Storhas (Bioreaktoren und periphere Einrichtungen (ViewegVerlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must satisfy in a suitable manner thedemands of the respective strains. There are descriptions of culturemedia for various microorganisms in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

These media which can be employed according to the invention usuallycomprise one or more carbon sources, nitrogen sources, inorganic salts,vitamins and/or trace elements.

Preferred carbon sources are sugars such as mono-, di- orpolysaccharides. Examples of very good carbon sources are glucose,fructose, mannose, galactose, ribose, ribose, sorbose, ribulose,lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars can beput in the media also via complex compounds such as molasses, or otherby-products of sugar refining. It may also be advantageous to addmixtures of various carbon sources. Other possible carbon sources areoils and fats such as, for example, soybean oil, sunflower oil, peanutoil and coconut fat, fatty acids such as, for example, palmitic acid,stearic acid or linoleic acid, alcohols such as, for example, glycerol,methanol or ethanol and organic acids such as, for example, acetic acidor lactic acid.

Nitrogen sources are usually organic or inorganic nitrogen compounds ormaterials containing these compounds. Examples of nitrogen sourcesinclude ammonia gas or ammonium salts such as ammonium sulfate, ammoniumchloride, ammonium phosphate, ammonium carbonate or ammonium nitrate,nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soybean flour, soybean protein, yeast extract, meatextract and others. The nitrogen sources may be used singly or asmixtures.

Inorganic salt compounds which may be present in the media comprise thechloride, phosphoric or sulfate salts of calcium, magnesium, sodium,cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

For producing fine chemicals, especially methionine, it is possible touse as sulfur source inorganic compounds such as, for example, sulfates,sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but alsoorganic sulfur compounds such as mercaptans and thiols.

It is possible to use as phosphorus source phosphoric acid, potassiumdihydrogenphosphate or dipotassium hydrogenphosphate or thecorresponding sodium-containing salts.

Chelating agents can be added to the medium in order to keep the metalions in solution. Particularly suitable chelating agents comprisedihydroxyphenols such as catechol or protocatechuate, or organic acidssuch as citric acid.

The fermentation media employed according to the invention normally alsocomprise other growth factors such as vitamins or growth promoters,which include for example biotin, riboflavin, thiamine, folic acid,nicotinic acid, pantothenate and pyridoxine.

Growth factors and salts are frequently derived from complex componentsof the media, such as yeast extract; molasses, corn steep liquor and thelike. Suitable precursors may also be added to the culture medium. Theexact composition of the compounds in the media depends greatly on theparticular experiment and will be decided individually for each specificcase. Information on optimization of media is obtainable from thetextbook “Applied Microbiol. Physiology, A Practical Approach” (editorsP. M. Rhodes, P. F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19963577 3). Growth media can also be purchased from commercial suppliers,such as Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) and thelike.

All the components of the media are sterilized either by heat (20 min at1.5 bar and 121° C.) or by sterilizing filtration. The components can besterilized either together or, if necessary, separately. All thecomponents of the media may be present at the start of culturing oroptionally be added continuously or batchwise.

The temperature of the culture is normally between 15° C. and 45° C.,preferably at 25° C. to 40° C. and can be kept constant or changedduring the experiment. The pH of the medium should be in the range from5 to 8.5, preferably around 7.0. The pH for the culturing can becontrolled during the culturing by adding basic compounds such as sodiumhydroxide, potassium hydroxide, ammonia or aqueous ammonia or acidiccompounds such as phosphoric acid or sulfuric acid. The development offoam can be controlled by employing antifoams such as, for example,fatty acid polyglycol esters. The stability of plasmids can bemaintained by adding to the medium suitable substances with a selectiveaction, such as, for example, antibiotics. Aerobic conditions aremaintained by introducing oxygen or oxygen-containing gas mixtures suchas, for example, ambient air into the culture. The temperature of theculture is normally 20° C. to 45° C. The culture is continued untilformation of the desired product is at a maximum. This aim is normallyreached within 10 hours to 160 hours.

The dry matter content of the fermentation broths obtained in this wayis normally from 7.5 to 25% by weight.

It is additionally advantageous also to run the fermentation with sugarlimitation at least at the end, but in particular over at least 30% ofthe fermentation time. This means that the concentration of utilizablesugar in the fermentation medium is kept at 0 to 3 g/l, or is reduced,during this time.

Biosynthetic products are isolated from the fermentation broth and/orthe microorganisms in a manner known per se in accordance with thephysical/chemical properties of the required biosynthetic product andthe biosynthetic by-products.

The fermentation broth can then be processed further for example.Depending on the requirement, the biomass can be removed wholly orpartly from the fermentation broth by separation methods such as, forexample, centrifugation, filtration, decantation or a combination ofthese methods, or left completely in it.

The fermentation broth can then be concentrated by known methods suchas, for example, with the aid of a rotary evaporator, thin-filmevaporator, falling-film evaporator, by reverse osmosis or bynanofiltration. This concentrated fermentation broth can then be workedup by freeze drying, spray drying, spray granulation or by othermethods.

However, it is also possible to purify the biosynthetic products,especially L-lysine, L-methionine and L-threonine, further. For thispurpose, the product-containing broth is subjected, after removal of thebiomass, to a chromatography using a suitable resin, with the desiredproduct or the impurities being retained wholly or partly on thechromatography resin. These chromatographic steps can be repeated ifrequired, using the same or different chromatography resins. The skilledworker is proficient in the selection of suitable chromatography resinsand their most effective use. The purified product can be concentratedby filtration or ultrafiltration and be stored at a temperature at whichthe stability of the product is a maximum.

The biosynthetic products may result in various forms, for example inthe form of their salts or esters.

The identity and purity of the isolated compound(s) can be determined byprior art techniques. These comprise high performance liquidchromatography (HPLC), spectroscopic methods, staining methods,thin-layer chromatography, NIRS, enzyme assay or microbiological assays.These analytical methods are summarized in: Patek et al. (1994) Appl.Environ. Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya11 27-32; and Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.Ulmann's Encyclopedia of Industrial Chemistry (1996) vol. A27, VCH:Weinheim, pp. 89-90, pp. 521-540, pp. 540-547, pp. 559-566, 575-581 andpp. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas ofBiochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. etal. (1987) Applications of HPLC in Biochemistry in: LaboratoryTechniques in Biochemistry and Molecular Biology, vol. 17.

The invention is now described in more detail by means of the followingnonlimiting examples:

EXAMPLE 1 Construction of Plasmid pCIS lysC

In the first step of strain construction, an allelic exchange of thelysC wild-type gene in Corynebacterium glutamicum ATCC13032 was carriedout. In this case, a nucleotide exchange was carried out in the lysCgene so that in the resulting protein the amino acid Thr at position 311was replaced by an Ile. Starting from the chromosomal DNA from ATCC13032as template for a PCR reaction, lysC was amplified with theoligonucleotide primers SEQ ID NO:5 and SEQ ID NO:6 with the aid of thePfu-Turbo PCR system (Stratagene USA) in accordance with themanufacturer's instructions.

SEQ ID NO:5 5′-GAGAGAGAGACGCGTCCCAGTGGCTGAGACGCATC-3′ SEQ ID NO:65′-CTCTCTCTGTCGACGAATTCAATCTTACGGCCTG-3′

Chromosomal DNA was prepared from C. glutamicum ATCC 13032 as describedby Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828. The amplified fragment is flanked at its 5′end by a SalI restriction site and at its 3′ end by a MluI restrictionsite. Before the cloning, the amplified fragment was digested with thesetwo restriction enzymes and purified using GFX™PCR, DNA and Gel Bandpurification kit (Amersham Pharmacia, Freiburg).

The resulting polynucleotide was cloned via the SalI and MluIrestriction sites into pCLIK5 MCS integrative SacB called pCIShereinafter (SEQ ID NO: 7) and transformed into E. coli XL-1 blue.Selection for plasmid-harboring cells was achieved by plating out on LBagar (Lennox, 1955, Virology, 1:190) containing kanamycin (20 μg/ml).The plasmid was isolated and the expected nucleotide sequence wasconfirmed by sequencing. Preparation of the plasmid DNA was carried outby methods and with materials from Qiagen. Sequencing reactions werecarried out as described by Sanger et al. (1977) Proceedings of theNational Academy of Sciences USA 74:5463-5467. The sequencing reactionswere fractionated and evaluated using an ABI prism 377 (PE AppliedBiosystems, Weiterstadt). The resulting plasmid pCIS lysC is listed asSEQ ID NO:8.

EXAMPLE 2 Mutagenesis of the lysC gene from C. glutamicum

Directed mutagenesis of the lysC gene from C. glutamicum was carried outwith the Quickchange kit (from Stratagene/USA) in accordance with themanufacturer's instructions. The mutagenesis was carried out in theplasmid pCIS lysC, SEQ ID NO:8. For the exchange from thr 311 to 311 ileby means of the Quickchange method (Stratagene), the followingoligonucleotide primers were synthesized:

SEQ ID NO:9 5′-CGGCACCACCGACATCATCTTCACCTGCCCTCGTTCCG-3′ SEQ ID NO:105′-CGGAACGAGGGCAGGTGAAGATGATGTCGGTGGTGCCG-3′

Use of these oligonucleotide primers in the Quickchange reaction leadsto an exchange of the nucleotide in position 932 (from C to T) in thelysC gene SEQ ID NO: 11. The resulting amino acid exchange Thr311Ile inthe lysC gene was confirmed after transformation into E. coli XL1-blueand plasmid preparation by a sequencing reaction. The plasmid receivedthe designation pCIS lysC thr311 ile and is listed as SEQ ID NO:12.

The plasmid pCIS lysC thr311 ile was transformed into C. glutamicumATCC13032 by electroporation as described by Liebl et al. (1989) FEMSMicrobiology Letters 53:299-303. Modifications of the protocol aredescribed in DE 10046870. The chromosomal arrangement of the lysC locusof individual transformants was checked by Southern blotting andhybridization using standard methods as described in Sambrook et al.(1989), Molecular Cloning. A Laboratory Manual, Cold Spring Harbor. Thisensured that the transformants have integrated the transformed plasmidby homologous recombination at the lysC locus. After such colonies hadgrown overnight in media containing no antibody, the cells were platedout on a sucrose CM agar medium (10% sucrose, 10 g/l glucose; 2.5 g/lNaCl; 2 g/l urea, 10 g/l Bacto peptone (from Difco); 10 g/l yeastextract, 22.0 g/L agar (Difco)) and incubated at 30° C. for 24 hours.

Since the sacB gene present in the vector pCIS lysC thr311ile convertssucrose into a toxic product, the only colonies able to grow are thosewhich have deleted the sacB gene by a second homologous recombinationstep between the wild-type lysC gene and the mutated lysC thr311ilegene. During the homologous recombination there may be deletion eitherof the wild-type gene or of the mutated gene together with the sacBgene. Deletion of the SacB gene together with the wild-type gene resultsin a mutated transformant.

Grown colonies were picked and investigated for a kanamycin-sensitivephenotype. Clones with deleted SacB gene must simultaneously showkanamycin-sensitive growth behavior. Such Kan-sensitive clones wereinvestigated in a shaken flask for their lysine productivity (seeexample 6). The untreated C. glutamicum ATCC13032 was cultured forcomparison. Clones with lysine production increased compared with thecontrol were selected, chromosomal DNA isolated and the correspondingregion of the lysC gene amplified by a PCR reaction and sequenced. Sucha clone with the property of increased lysine synthesis and demonstratedmutation in lysC at position 932 was referred to as ATCC13032lySC^(fbr).

EXAMPLE 3 Preparation of an Integrated Plasmid for Overexpression of thelysC Gene with the Aid of the heterologous expression unit Psod (SEQ.ID. NO. 2)

The following oligonucleotides were defined for amplification of theexpression unit of the gene which codes for superoxide dismutase.

SEQ ID 13: sod8: 5′-accctggcggaaaccctgagtcg-3′ SEQ ID 14: sod1:5′-tacgaccagggccacgggtaaaaaatcctttcgtaggtttccgcac cgagcatatacatcttttg-3′

The primers were employed in a PCR reaction with chromosomal DNA from C.glutamicum ATCC 13032. It was possible with this approach to amplify aDNA fragment which corresponded to the expected size of 657 bp.

The following oligonucleotides were defined for amplification of thegene which codes for aspartokinase.

SEQ ID 15 lysC2: 5′-cctacgaaaggattttttacccgtggccctggtcgtacag-3′ SEQ ID16: lysC6: 5′-gattagtggaacctcgtcgtc-3′

The primers were employed in a PCR reaction with chromosomal DNA from C.glutamicum ATCC13032 lysC^(fbr). It was possible with this approach toamplify a DNA fragment which corresponded to the expected size of 1638bp.

The primers sod1 and ask2 contain an overlapping sequence and arehomologous to one another at their 5′ ends.

The PCR products obtained above were employed as template for a furtherPCR in which the following primers were used.

SEQ ID 17: sod4: 5′-gcggcgcaggattttctaa-3′ SEQ ID 18: lysC4:5′-tcggttgcctgagtaatgtctt-3′

It was possible with this approach to amplify a DNA fragment whichcorresponded to the expected size of 1811 bp. This Psod/lysC fusion wasthen cloned into the vector pCR2.1 (from Invitrogen GmbH, Karlsruhe,Germany). In a further step, the Psod/lysC fusion was cloned from theplasmid pCR2.1 (from Invitrogen GmbH, Karlsruhe, Germany) as 1773 bpEcoRI fragment into the integration vector pK19 mob sacB SEQ ID NO 19,which had previously been cut with the restriction endonuclease EcoRI.The resulting plasmid was referred to as pk19 mob sacB Psod lysC.

The following oligonucleotides were defined for amplification of a 5′region of the lysC gene:

SEQ ID 20: lysC23: 5′-caccgcggctttggacatcactgctac-3′ SEQ ID 21: lysC24:5′-cctggggctttagcggatgcgtctca-3′

The primers were employed in a PCR reaction with chromosomal DNA from C.glutamicum. It was possible with this approach to amplify a DNA fragmentwhich corresponded to the expected size of 674 bp. This DNA fragment wascloned into the vector pCR2.1 (from Invitrogen GmbH, Karlsruhe,Germany). A 787 bp SpeI/XbaI fragment was then subsequently cloned intothe vector pK19 mob sacB Psod lysC, which had previously been digestedwith the restriction enzyme NheI. The resulting plasmid was referred toas pK19 mob sacB Psod lysC+US (SEQ ID 22). Up to this step, all cloningswere carried out in Escherichia coli XL-1 Blue (from Stratagene,Amsterdam, Netherlands).

The transformation plasmid pK19 mob sacB Psod lysC+US was then used totransform E. coli Mn522 (from Stratagene, Amsterdam, Netherlands)together with the plasmid pTc15AcgIM as described by Liebl et al. (1989)FEMS Microbiology Letters 53:299-303. The plasmid pTc15AcgIM enables DNAto be methylated according to the methylation pattern of Corynebacteriumglutamicum (DE 10046870). This step enables Corynebacterium glutamicumsubsequently to undergo electroporation with the integration plasmidpK19 mob sacB Psod lysC+US. This electroporation and the subsequentselection on CM plates with kanamycin (25 μg/ml) resulted in a pluralityof transconjugants. To select for the second recombination event, whichshould lead to excision of the vector together with the lysC promoterand the lysC gene, these transconjugants were cultured in CM mediumwithout kanamycin overnight and then plated out on CM plates with 10%sucrose for selection. The sacB gene present on the vector pK19 mob sacBcodes for the enzyme levansucrase and leads to the synthesis of levan ongrowth on sucrose. Since levan is toxic for C. glutamicum, the only C.glutamicum cells able to grow on sucrose-containing medium are thosewhich have lost the integration plasmid through the second recombinationstep (Jäger et al., Journal of Bacteriology 174 (1992) 5462-5466). 100sucrose-resistant clones were examined for their kanamycin sensitivity.It was possible to demonstrate for 57 of the tested clones not onlyresistance to sucrose but also sensitivity to kanamycin. A polymerasechain reaction (PCR) was used to check whether the desired replacementof the natural expression unit by the Psod expression unit had alsotaken place. Chromosomal DNA was isolated from the initial strain and 20clones for this analysis. For this purpose, the respective clones wereremoved from the agar plate with a toothpick and suspended in 100 μl ofH₂O and boiled at 95° C. for 10 min. 10 μl portions of the resultingsolution were employed as template in the PCR. The primers used wereoligonucleotides which are homologous to the Psod expression unit andthe lysC gene. The PCR conditions were chosen as follows:predenaturation: 5 min at 95° C.; denaturation 30 sec at 95° C.;hybridization 30 sec at 55° C.; amplification 2 min at 72° C.; 30cycles; final extension 5 min at 72° C. In the mixture with the DNA ofthe initial strain it was not possible for a PCR product to result owingto the choice of the oligonucleotides. Only with clones in which thesecond recombination effected replacement of the natural promoter(PlysC) by Psod was a band with a size of 340 bp expected. In total, 2of the tested 20 clones were positive.

EXAMPLE 4 Aspartokinase (lysC) Assay

C. glutamicum strains which contained either a chromosomal copy of thelysC^(fbr) gene with the natural promoter or a chromosomal copy of thePsod lysC^(fbr) construct were cultured in MMA medium (20 g/l glucose,10 g/l (NH₄)₂SO₄*0.5 g KH₂PO₄*0.5 g K₂HPO₄*2.5 g urea, 5 g NaCl, 400 mgMgSO₄.7H₂O, 10 mg FeSO₄*7H₂O, MnSO₄*6H₂O, 100 mg L-leucine, 100 mgL-cysteine, 250 mg pantothenic acid, 5 mg nicotinamide, 100 μg biotin,200 μg thiamine, 15 g MOPS, pH 6.8) at 30° C. until the OD₆₀₀ was 8. Thecells were spun down at 4° C. and then washed twice with cold tris-HClbuffer (0.1%, pH 8.0). After renewed centrifugation, the cells weretaken up in cold tris-HCl buffer (0.1%, pH 8.0) and adjusted to an OD₆₀₀of 160. For cell disruption, 1 ml of this cell suspension wastransferred into 2 ml Ribolyser tubes from Hybaid and lysed in aRibolyser from Hybaid at a rotation setting of 6.0 three times for 30sec each time. The lysate was clarified by centrifugation at 15 000 rpmand 4° C. in an Eppendorf centrifuge for 30 minutes, and the supernatantwas transferred into a new Eppendorf cup. The protein content wasdetermined as described by Bradford, M. M. (1976) Anal. Biochem.72:248-254.

The aspartokinase enzymatic activity was determined as follows. 1 mlreaction mixtures with 100 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 600 mMhydroxylamine HCl (pH 7.0 with 10N KOH), 4 mM ATP, 200 mM aspartate(sodium salt) and H₂O ad 1 ml were incubated at 30° C. for 10 min. Theassay was started by adding the respective protein lysate and inubatingat 30° C. for 30 min. The reaction was stopped by adding 1 ml of thestop solution (10% iron chloride, 3.3% trichloroacetic acid, 0.7N NaCl)to the reaction mixture. After a centrifugation step, the OD₅₄₀ of thesupernatant was measured. 1 unit in this case is equivalent to 1 nmol ofaspartate hydroxamate formed per mg of protein and per minute.

The results are shown in Table 1a.

TABLE 1a Specific activity Strain [nmol/mg/min] ATCC 13032 lysC^(fbr)19.35 ATCC 13032 Psod lysC^(fbr) 41.22

It was possible to double the aspartokinase activity by integrating thePsod lysC contruct into the chromosome.

EXAMPLE 5 Production of Lysine

To investigate the effect of the Psod lysC construct on lysineproduction, the strain ATCC13032, ATCC13032 lysC^(fbr) or ATCC13032 PsodlySC^(fbr) was cultured on CM plates (10.0 g/l D-glucose, 2.5 g/l NaCl,2.0 g/l urea, 10.0 g/l Bacto peptone (Difco), 5.0 g/l yeast extract(Difco), 5.0 g/l beef extract (Difco), 22.0 g/l agar (Difco), autoclaved(20 min. 121° C.)) at 30° C. for 2 days. The cells were then scraped offthe plate and resuspended in saline. For the main culture, 10 ml ofmedium 1 and 0.5 g of autoclaved CaCO₃ (Riedel de Haen) in a 100 mlErlenmeyer flask were inoculated with the cell suspension until theOD₆₀₀ was 1.5 and incubated on a shaker of the type Infors AJ118 (fromInfors, Bottmingen, Switzerland) at 220 rpm for 39 h. The concentrationof the lysine secreted into the medium was then determined.

Medium I:

40 g/l sucrose60 g/l molasses (calculated for 100% sugar content)

10 g/l (NH₄)₂SO₄ 0.4 g/l MgSO₄*7H₂O 0.6 g/l KH₂PO₄

0.3 mg/l thiamin*HCl1 mg/l biotin (from a 1 mg/ml stock solution which had been sterilizedby filtration and adjusted to pH 8.0 with NH₄OH)2 mg/l FeSO₄2 mg/l MnSO₄adjusted to pH 7.8 with NH₄OH, autoclaved (121° C., 20 min).

In addition, vitamin B12 (hydroxycobalamin Sigma Chemicals) from a stocksolution (200 μg/ml, sterilized by filtration) is added to a finalconcentration of 100 μg/l.

The amino acid concentration was determined by Agilent high pressureliquid chromatography on an Agilent 1100 series LC system HPLC.Precolumn derivatization with ortho-phthalaldehyde permitsquantification of the amino acids formed, and the amino acid mixture isfractionated on a Hypersil AA column (Agilent). The result of theinvestigation is shown in Table 2a.

TABLE 2a Strain L-lysine (g/l) ATCC13032 0 ATCC13032 lysC^(fbr) 10.15ATCC13032 Psod lysC^(fbr) 12.67

EXAMPLE 6 Preparation of the Vector pCLiK5MCS

Firstly, the ampicillin resistance and origin of replication of thevector pBR322 were amplified by the polymerase chain reaction (PCR)using the oligonucleotide primers SEQ ID NO: 23 and SEQ ID NO: 24.

SEQ ID NO: 23 5′-CCCGGGATCCGCTAGCGGCGCGCCGGCCGGCCCGGTGTGAAATACCGCACAG-3′ SEQ ID NO: 245′-TCTAGACTCGAGCGGCCGCGGCCGGCCTTTAAATTGAAGACGAAAGG GCCTCG-3′

Besides the sequences complementary to pBR322, the oligonucleotideprimer SEQ ID NO: 23 contains in the 5′-3′ direction the cleavage sitesfor the restriction endonucleases SmaI, BamHI, NheI and AscI and theoligonucleotide primer SEQ ID NO: 24 contains in the 5′-3′ direction thecleavage sites for the restriction endonucleases XbaI, XhoI, NotI andDraI. The PCR reaction was carried out with PfuTurbo polymerase(Stratagene, La Jolla, USA) by a standard method such as Innis et al.(PCR Protocols. A Guide to Methods and Applications, Academic Press(1990)). The resulting DNA fragment with a size of approximately 2.1 kbas purified using the GFX™PCR, DNA and Gel Band purification kit(Amersham Pharmacia, Freiburg) in accordance with the manufacturer'sinstructions. The blunt ends of the DNA fragment were ligated togetherusing the rapid DNA ligation kit (Roche Diagnostics, Mannheim) inaccordance with the manufacturer's instructions, and the ligationmixture was transformed into competent E. coli XL-1 Blue (Stratagene, LaJolla, USA) by standard methods as described in Sambrook et al.(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor (1989)).Plasmid-harboring cells were selected by plating out on LB agar (Lennox,1955, Virology, 1:190) containing ampicillin (50 μg/ml).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) in accordance with the manufacturer'sinstructions and checked by restriction digestions. The plasmid obtainedin this way is called pCLiK1.

Starting from the plasmid pWLT1 (Liebl et al., 1992) as template for aPCR reaction, a kanamycin resistance cassette was amplified using theoligonucleotide primers SEQ ID NO: 25 and SEQ ID NO: 26.

SEQ ID NO: 25: 5′-GAGATCTAGACCCGGGGATCCGCTAGCGGGCTGCTAAAGGAAGCGG A-3′SEQ ID NO: 26 5′-GAGAGGCGCGCCGCTAGCGTGGGCGAAGAACTCCAGCA-3′

Besides the sequences complementary to pWLT1, the oligonucleotide primerSEQ ID NO: 25 contains in the 5′-3′ direction the cleavage sites for therestriction endonucleases XbaI, SmaI, BamHI, NheI and theoligonucleotide primer SEQ ID NO: 26 contains in the 5′-3′ direction thecleavage sites for the restriction endonucleases AscI and NheI. The PCRreaction was carried out with PfuTurbo polymerase (Stratagene, La Jolla,USA) by standard methods such as Innis et al. (PCR

Protocols. A Guide to Methods and Applications, Academic Press (1990)).The resulting DNA fragment with a size of approximately 1.3 kb waspurified using the GFX™PCR, DNA and Gel Band purification kit (AmershamPharmacia, Freiburg) in accordance with the manufacturer's instructions.The DNA fragment was cut with the restriction endonucleases XbaI andAscI (New England Biolabs, Beverly, USA) and subsequently again purifiedusing the GFX™PCR, DNA and Gel Band purification kit (AmershamPharmacia, Freiburg) in accordance with the manufacturer's instructions.The vector pCLiK1 was likewise cut with the restriction endonucleasesXbaI and AscI and dephosphorylated with alkaline phosphatase (I (RocheDiagnostics, Mannheim)) in accordance with the manufacturer'sinstructions. After electrophoresis in a 0.8% agarose gel, thelinearized vector (approx. 2.1 kb) was isolated using the GFX™PCR, DNAand Gel Band purification kit (Amersham Pharmacia, Freiburg) inaccordance with the manufacturer's instructions. This vector fragmentwas ligated with the cut PCR fragment using the rapid DNA ligation kit(Roche Diagnostics, Mannheim) in accordance with the manufacturer'sinstructions, and the ligation mixture was transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA) by standard methods asdescribed in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor, (1989)). Plasmid-harboring cells were selected byplating out on LB agar (Lennox, 1955, Virology, 1:190) containingampicillin (50 μg/ml) and kanamycin (20 μg/ml).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) in accordance with the manufacturer'sinstructions and checked by restriction digestions. The plasmid obtainedin this way is called pCLiK2.

The vector pCLiK2 was cut with the restriction endonuclease DraI (NewEngland Biolabs, Beverly, USA). After electrophoresis in a 0.8% agarosegel, a vector fragment approx. 2.3 kb in size was isolated using theGFX™PCR, DNA and Gel Band purification kit (Amersham Pharmacia,Freiburg) in accordance with the manufacturer's instructions. Thisvector fragment was religated using the rapid DNA ligation kit (RocheDiagnostics, Mannheim) in accordance with the manufacturer'sinstructions, and the ligation mixture was transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA) by standard methods asdescribed in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor, (1989)). Plasmid-harboring cells were selected byplating out on LB agar (Lennox, 1955, Virology, 1:190) containingkanamycin (20 μg/ml).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) in accordance with the manufacturer'sinstructions and checked by restriction digestions. The plasmid obtainedin this way is called pCLiK3.

Starting from the plasmid pWLQ2 (Liebl et al., 1992) as template for aPCR reaction, the origin of replication pHM1519 was amplified using theoligonucleotide primers SEQ ID NO: 27 and SEQ ID NO: 28.

SEQ ID NO: 27: 5′-GAGAGGGCGGCCGCGCAAAGTCCCGCTTCGTGAA-3′ SEQ ID NO: 28:5′-GAGAGGGCGGCCGCTCAAGTCGGTCAAGCCACGC-3′

Besides the sequences complementary to pWLQ2, the oligonucleotideprimers SEQ ID NO: 27 and SEQ ID NO: 28 contain cleavage sites for therestriction endonuclease NotI. The PCR reaction was carried out withPfuTurbo polymerase (Stratagene, La Jolla, USA) by a standard methodsuch as Innis et al. (PCR Protocols. A Guide to Methods andApplications, Academic Press (1990)). The resulting DNA fragment with asize of approximately 2.7 kb was purified using the GFX™PCR, DNA and GelBand purification kit (Amersham Pharmacia, Freiburg) in accordance withthe manufacturer's instructions. The DNA fragment was cut with therestriction endonuclease NotI (New England Biolabs, Beverly, USA) andthen again purified with the GFX™PCR, DNA and Gel Band purification kit(Amersham Pharmacia, Freiburg) in accordance with the manufacturer'sinstructions. The vector pCLiK3 was likewise cut with the restrictionendonuclease NotI and dephosphorylated with alkaline phosphatase (I(Roche Diagnostics, Mannheim)) in accordance with the manufacturer'sinstructions. After electrophoresis in a 0.8% agarose gel, thelinearized vector (approx. 2.3 kb) was isolated with the GFX™PCR, DNAand Gel Band purification kit (Amersham Pharmacia, Freiburg) inaccordance with the manufacturer's instructions. This vector fragmentwas ligated with the cut PCR fragment using the rapid DNA ligation kit(Roche Diagnostics, Mannheim) in accordance with the manufacturer'sinstructions, and the ligation mixture was transformed into competent E.coli XL-1 Blue (Stratagene, La Jolla, USA) by standard methods asdescribed in Sambrook et al. (Molecular Cloning. A Laboratory Manual,Cold Spring Harbor, (1989)). Plasmid-harboring cells were selected byplating out on LB agar (Lennox, 1955, Virology, 1:190) containingkanamycin (20 μg/ml).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) in accordance with the manufacturer'sinstructions and checked by restriction digestions. The plasmid obtainedin this way is called pCLiK5. To extend pCLiK5 by a multiple cloningsite (MCS), the two synthetic, very substantially complementaryoligonucleotides SEQ ID NO: 29 and SEQ ID NO: 30, which contain cleavagesites for the restriction endonucleases SwaI, XhoI, AatI, ApaI, Asp718,MluI, NdeI, SpeI, EcoRV, SalI, ClaI, BamHI, XbaI and SmaI, were combinedby heating together at 95° C. and slow cooling to give a double-strandedDNA fragment.

SEQ ID NO: 29: 5′-TCGAATTTAAATCTCGAGAGGCCTGACGTCGGGCCCGGTACCACGCGTCATATGACTAGTTCGGACCTAGGGATATCGTCGACATCGATGCTCTTCTGCGTTAATTAACAATTGGGATCCTCTAGACCCGGGATTTAAAT-3′ SEQ ID NO: 30:5′-GATCATTTAAATCCCGGGTCTAGAGGATCCCAATTGTTAATTAACGCAGAAGAGCATCGATGTCGACGATATCCCTAGGTCCGAACTAGTCATATGACGCGTGGTACCGGGCCCGACGTCAGGCCTCTCGAGATTTAAAT-3′

The vector pCLiK5 was cut with the restriction endonucleases XhoI andBamHI (New England Biolabs, Beverly, USA) and dephosphorylated withalkaline phosphatase (I (Roche Diagnostics, Mannheim)) in accordancewith the manufacturer's instructions. After electrophoresis in a 0.8%agarose gel, the linearized vector (approx. 5.0 kb) was isolated withthe GFX™PCR, DNA and Gel Band purification kit (Amersham Pharmacia,Freiburg) in accordance with the manufacturer's instructions. Thisvector fragment was ligated to the synthetic double-stranded DNAfragment using the rapid DNA ligation kit (Roche Diagnostics, Mannheim)in accordance with the manufacturer's instructions, and the ligationmixture was transformed into competent E. coli XL-1 Blue (Stratagene, LaJolla, USA) by standard methods as described in Sambrook et al.(Molecular Cloning. A Laboratory Manual, Cold Spring Harbor, (1989)).Plasmid-harboring cells were selected by plating out on LB agar (Lennox,1955, Virology, 1:190) containing kanamycin (20 μg/ml).

The plasmid DNA of an individual clone was isolated using the Qiaprepspin miniprep kit (Qiagen, Hilden) in accordance with the manufacturer'sinstructions and checked by restriction digestions. The plasmid obtainedin this way is called pCLiK5MCS.

Sequencing reactions were carried out as described by Sanger et al.(1977) Proceedings of the National Academy of Sciences USA 74:5463-5467.The sequencing reactions were fractionated and evaluated using an ABIprism 377 (PE Applied Biosystems, Weiterstadt).

The resulting plasmid pCLiK5MCS is listed as SEQ ID NO: 31.

EXAMPLE 7 Preparation of the Plasmid PmetA metA

Chromosomal DNA was prepared from C. glutamicum ATCC 13032 as describedby Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828. The meta gene including the noncoding 5′region was amplified by the polymerase chain reaction (PCR) by standardmethods as described in Innis et al. (1990) PCR Protocols. A Guide toMethods and Applications, Academic Press, using the oligonucleotideprimers SEQ ID NO: 32 and SEQ ID NO: 33, the chromosomal DNA as templateand Pfu Turbo polymerase (from Stratagene).

5′-GCGCGGTACCTAGACTCACCCCAGTGCT-3′ SEQ ID NO: 32 and5′-CTCTACTAGTTTAGATGTAGAACTCGATGT-3′ SEQ ID NO: 33

The resulting DNA fragment with a size of approx. 1.3 kb was purifiedusing the GFX™PCR, DNA and Gel Band purification kit (AmershamPharmacia, Freiburg) in accordance with the manufacturer's instructions.It was then cleaved with the restriction enzymes Asp718 and SpeI (RocheDiagnostics, Mannheim) and the DNA fragment was purified with theGFX™PCR, DNA and Gel Band purification kit.

The vector pClik5MCS SEQ ID NO: 31 was cut with the restriction enzymesAsp718 and SpeI and, after fractionation by electrophoresis, a fragment5 kb in size was isolated using the GFX™PCR, DNA and Gel Bandpurification kit.

The vector fragment was ligated together with the PCR fragment using therapid DNA ligation kit (Roche Diagnostics, Mannheim) in accordance withthe manufacturer's instructions, and the ligation mixture wastransformed into competent E. coli XL-1 Blue (Stratagene, La Jolla, USA)by standard methods as described in Sambrook et al. (Molecular Cloning.A Laboratory Manual, Cold Spring Harbor, (1989)). Plasmid-harboringcells were selected by plating out on LB agar (Lennox, 1955, Virology,1:190) containing kanamycin (20 μg/ml).

The plasmid DNA was prepared by methods and using materials from Qiagen.Sequencing reactions were carried out as described by Sanger et al.(1977) Proceedings of the National Academy of Sciences USA 74:5463-5467.The sequencing reactions were fractionated and evaluated using an ABIprism 377 (PE Applied Biosystems, Weiterstadt).

The resulting plasmid pCLiK5MCS PmetA meta is listed as SEQ ID NO: 34.

EXAMPLE 8 Preparation of the Plasmid pCLiK5MCS Psod metA

Chromosomal DNA was prepared from C. glutamicum ATCC 13032 as describedby Tauch et al. (1995) Plasmid 33:168-179 or Eikmanns et al. (1994)Microbiology 140:1817-1828. A DNA fragment of approx. 200 base pairsfrom the noncoding 5′ region (region of the expression unit) ofsuperoxide dismutase (Psod) was amplified by the polymerase chainreaction (PCR) by standard methods such as Innis et al. (1990) PCRProtocols. A Guide to Methods and Applications, Academic Press, usingthe oligonucleotide primers SEQ ID NO: 35 and SEQ ID NO: 36, thechromosomal DNA as template and Pfu Turbo polymerase (from Stratagene).

SEQ ID NO: 35 5′-GAGACTCGAGAGCTGCCAATTATTCCGGG-3′ and SEQ ID NO: 365′-CCTGAAGGCGCGAGGGTGGGCATGGGTAAAAAATCCTTTCG-3′

The resulting DNA fragment was purified with the GFX™PCR, DNA and GelBand purification kit (Amersham Pharmacia, Freiburg) in accordance withthe manufacturer's instructions.

Starting from plasmid PmetA meta SEQ ID 34 as template for a PCRreaction, a part of meta was amplified using the oligonucleotide primersSEQ ID NO: 37: and SEQ ID NO: 38.

5′-CCCACCCTCGCGCCTTCAG-3′ SEQ ID NO: 37 and 5′-CTGGGTACATTGCGGCCC-3′ SEQID NO: 38

The resulting DNA fragment of approximately 470 base pairs was purifiedwith the GFX™PCR, DNA and Gel Band purification kit in accordance withthe manufacturer's instructions.

In a further PCR reaction, the two fragments obtained above wereemployed together as template. Owing to the sequences which have beenintroduced with the oligonucleotide primer SEQ ID NO: 36 and arehomologous to meta, during the PCR reaction the two fragments areattached to one another and extended to give a continuous DNA strand bythe polymerase employed. The standard method was modified by adding theoligonucleotide primers used SEQ ID NO: 35 and SEQ ID NO: 38, to thereaction mixture only at the start of the second cycle.

The amplified DNA fragment of approximately 675 base pairs was purifiedusing the GFX™PCR, DNA and Gel Band purification kit in accordance withthe manufacturer's instructions. It was then cleaved with therestriction enzymes XhoI and NcoI (Roche Diagnostics, Mannheim) andfractionated by gel electrophoresis. Subsequently, the DNA fragmentapproximately 620 base pairs in size was purified from the agarose usingthe GFX™PCR, DNA and Gel Band purification kit (Amersham Pharmacia,Freiburg). The plasmid PmetA meta SEQ ID NO: 34 was cleaved with therestriction enzymes NcoI and SpeI (Roche Diagnostics, Mannheim). Afterfractionation by gel electrophoresis, a meta fragment approximately 0.7kb in size was purified from the agarose using the GFX™PCR, DNA and GelBand purification kit.

The vector pClik5MCS SEQ ID NO: 31 was cut with the restriction enzymesXhoI and SpeI (Roche Diagnostics, Mannheim) and, after fractionation byelectrophoresis, a fragment 5 kb in size was isolated using the GFX™PCR,DNA and Gel Band purification kit.

The vector fragment was ligated together with the PCR fragment and themeta fragment using the rapid DNA ligation kit (Roche Diagnostics,Mannheim) in accordance with the manufacturer's instructions, and theligation mixture was transformed into competent E. coli XL-1 Blue(Stratagene, La Jolla, USA) by standard methods as described in Sambrooket al. (Molecular Cloning. A Laboratory Manual, Cold Spring Harbor,(1989)). Plasmid-harboring cells were selected by plating out on LB agar(Lennox, 1955, Virology, 1:190) containing kanamycin (20 μg/ml).

The plasmid DNA was prepared by methods and using materials from Qiagen.Sequencing reactions were carried out as described by Sanger et al.(1977) Proceedings of the National Academy of Sciences USA 74:5463-5467.The sequencing reactions were fractionated and evaluated using an ABIprism 377 (PE Applied Biosystems, Weiterstadt).

The resulting plasmid pCLiK5MCS PSODmetA is listed as SEQ ID NO: 39.

EXAMPLE 9 MetA Activities

The strain Corynebacterium glutamicum ATCC13032 was transformed witheach of the plasmids pClik5 MCS, pClik MCS PmetA meta, pCLiK5MCS Psodmeta by the method described (Liebl, et al. (1989) FEMS MicrobiologyLetters 53:299-303). The transformation mixture was plated on CM plateswhich additionally contained 20 mg/l kanamycin in order to select forplasmid-containing cells. Resulting Kan-resistant clones were picked andisolated. C. glutamicum strains which contained one of these plasmidconstructs were cultured in MMA medium (40 g/l sucrose, 20 g/l(NH₄)₂SO₄, 1 g/l KH₂PO₄, 1 g/l K₂HPO₄, 0.25 g/l MgSO₄×7H₂O, 54 g Aces, 1ml CaCl₂ (10 g/l), 1 ml protocatechuate (300 mg/10 ml), 1 ml traceelement solution (10 g/l FeSO₄×7H₂O, 10 g/l MnSO₄×H₂O, 2 g/l ZnSO₄×7H₂O,0.2 g/l CuSO₄, 0.02 g/l NiCl₂×6H₂O), 100 μg/l vitamin B₁₂, 0.3 mg/lthiamine, 1 mM leucine, 1 mg/l pyridoxal HCl, 1 ml biotin (100 mg/l), pH7.0) at 30° C. overnight. The cells were spun down at 4° C. and thenwashed twice with cold Tris-HCl buffer (0.1%, pH 8.0). After renewedcentrifugation, the cells were taken up in cold Tris-HCl buffer (0.1%,pH 8.0) and adjusted to an OD₆₀₀ of 160. For cell disruption, 1 ml ofthis cell suspension was transferred into 2 ml Ribolyser tubes fromHybaid and lysed in a Ribolyser from Hybaid with a rotation setting of6.0 three times for 30 sec each time. The lysate was clarified bycentrifugation at 15 000 rpm and 4° C. in an Eppendorf centrifuge for 30minutes, and the supernatant was transferred into a new Eppendororf cup.The protein content was determined as described by Bradford, M. M.(1976) Anal. Biochem. 72:248-254.

The measurement of the enzymatic activity of meta was carried out asfollows. The 1 ml reaction mixtures contained 100 mM potassium phosphatebuffer (pH 7.5), 5 mM MgCl₂, 100 μM acetyl-CoA, 5 mM L-homoserine, 500μM DTNB (Ellman's reagent) and cell extract. The assay was started byadding the respective protein lysate and incubated at room temperature.Kinetics were then recorded at 412 nm for 10 min.

The results are shown in Table 3a.

TABLE 3a Specific activity Strain [nmol/mg/min] ATCC 13032 pClik5MCS12.6 ATCC 13032 pClik5MCS PmetA metA 50.7 ATCC 13032 pClik5MCS Psod metA100.7

It was possible to increase MetA activity considerably by using theheterologous expression unit Psod.

1. A method of regulating the transcription of a gene comprisingintroducing into a host cell the nucleic acid molecule of claim 5 or anucleic acid molecule consisting of SEQ ID NO: 1, wherein the nucleicacid molecule has promoter activity.
 2. A method of regulating theexpression of a gene comprising introducing into a host cell theexpression unit of claim 6 or an expression unit consisting of SEQ IDNO:2.
 3. (canceled)
 4. (canceled)
 5. An isolated nucleic acid moleculehaving promoter activity, selected from the group consisting of havingpromoter activity, comprising A) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO:1; B) a nucleic acid moleculecomprising a nucleotide sequence of at least 90% identity to thenucleotide sequence of SEQ ID NO: 1; C) a nucleic acid molecule whichhybridizes with the complement of the nucleotide sequence of SEQ ID NO:1; D) a nucleic acid molecule comprising a fragment of the nucleic acidmolecule of (A), (B) or (C), wherein the molecule has promoter activity;wherein the nucleic acid molecule does not consist of SEQ ID NO:
 1. 6.An expression unit comprising a nucleic acid molecule having promoteractivity according to claim 5, wherein said nucleic acid molecule isfunctionally linked to a nucleic acid sequence which ensures thetranslation of ribonucleic acids.
 7. An expression unit according toclaim 6, comprising an isolated nucleic acid molecule selected from thegroup consisting of: E) a nucleic acid molecule comprising thenucleotide sequence of SEQ ID NO: 2; F) a nucleic acid moleculecomprising a nucleotide sequence of at least 90% identity to thenucleotide sequence of SEQ ID NO:2; G) a nucleic acid molecule whichhybridizes with the complement of the nucleotide sequence of SEQ IDNO:2; and H) a nucleic acid molecule comprising a fragment of thenucleic acid molecule of (E), (F) or (G), wherein the molecule hasexpression activity; wherein the nucleic acid molecule does not consistof SEQ ID NO:2.
 8. A method for altering or causing the transcriptionrate of genes in microorganisms compared with the wild type by a)altering the specific promoter activity in the microorganism ofendogenous nucleic acids having promoter activity according to claim 1,which regulate the transcription of endogenous genes, compared with thewild type or b) regulating the transcription of genes in themicroorganism by nucleic acids having promoter activity according toclaim 1 or by nucleic acids having promoter activity according to claim1 with altered specific promoter activity according to embodiment a),where the genes are heterologous in relation to the nucleic acids havingpromoter activity.
 9. The method according to claim 8, wherein theregulation of the transcription of genes in the microorganism by nucleicacids having promoter activity according to claim 1 or by nucleic acidshaving promoter activity according to claim 1 with altered specificpromoter activity according to embodiment a) is achieved by b1)introducing one or more nucleic acids having promoter activity accordingto claim 1, where appropriate with altered specific promoter activity,into the genome of the microorganism so that transcription of one ormore endogenous genes takes place under the control of the introducednucleic acid having promoter activity according to claim 1, whereappropriate with altered specific promoter activity, or b2) introducingone or more genes into the genome of the microorganism so thattranscription of one or more of the introduced genes takes place underthe control of the endogenous nucleic acids having promoter activityaccording to claim 1, where appropriate with altered specific promoteractivity, or b3) introducing one or more nucleic acid constructscomprising a nucleic acid having promoter activity according to claim 1,where appropriate with altered specific promoter activity, andfunctionally linked one or more nucleic acids to be transcribed, intothe microorganism.
 10. The method according to claim 8 or 9, wherein toincrease or cause the transcription rate of genes in microorganismscompared with the wild type ah) the specific promoter activity in themicroorganism of endogenous nucleic acids having promoter activityaccording to claim 1, or which regulate the transcription of endogenousgenes, is increased compared with the wild type, or bh) thetranscription of genes in the microorganism is regulated by nucleicacids having promoter activity according to claim 1 or by nucleic acidshaving increased specific promoter activity according to embodiment a),where the genes are heterologous in relation to the nucleic acids havingpromoter activity.
 11. The method according to claim 10, wherein theregulation of the transcription of genes in the microorganism by nucleicacids having promoter activity according to claim 1 or by nucleic acidshaving promoter activity according to claim 1 with increased specificpromoter activity according to embodiment a) is achieved by bh1)introducing one or more nucleic acids having promoter activity accordingto claim 1, where appropriate with increased specific promoter activity,into the genome of the microorganism so that transcription of one ormore endogenous genes takes place under the control of the introducednucleic acid having promoter activity according to claim 1, whereappropriate with increased specific promoter activity, or bh2)introducing one or more genes into the genome of the microorganism sothat transcription of one or more of the introduced genes takes placeunder the control of the endogenous nucleic acids having promoteractivity according to claim 1, where appropriate with increased specificpromoter activity, or bh3) introducing one or more nucleic acidconstructs comprising a nucleic acid having promoter activity accordingto claim 1, where appropriate with increased specific promoter activity,and functionally linked one or more nucleic acids to be transcribed,into the microorganism.
 12. The method according to claim 8 or 9,wherein to reduce the transcription rate of genes in microorganismscompared with the wild type ar) the specific promoter activity in themicroorganism of endogenous nucleic acids having promoter activityaccording to claim 1, which regulate the transcription of endogenousgenes, is reduced compared with the wild type, or br) nucleic acidshaving reduced specific promoter activity according to embodiment a) areintroduced into the genome of the microorganism so that thetranscription of endogenous genes takes place under the control of theintroduced nucleic acid having reduced promoter activity.
 13. A methodfor altering or causing the expression rate of a gene in microorganismscompared with the wild type by c) altering the specific expressionactivity in the microorganism of endogenous expression units accordingto claim 2, which regulate the expression of the endogenous genes,compared with the wild type or d) regulating the expression of genes inthe microorganism by expression units according to claim 2 or byexpression units according to claim 2 with altered specific expressionactivity according to embodiment c), where the genes are heterologous inrelation to the expression units.
 14. The method according to claim 13,wherein the regulation of the expression of genes in the microorganismby expression units according to claim 2 or by expression unitsaccording to claim 2 with altered specific expression activity accordingto embodiment a) is achieved by d1) introducing one or more expressionunits according to claim 2, where appropriate with altered specificexpression activity, into the genome of the microorganism so thatexpression of one or more endogenous genes takes place under the controlof the introduced expression units, or d2) introducing one or more genesinto the genome of the microorganism so that expression of one or moreof the introduced genes takes place under the control of the endogenousexpression units according to claim 2, where appropriate with alteredspecific expression activity, or d3) introducing one or more nucleicacid constructs comprising an expression unit according to claim 2,where appropriate with altered specific expression activity, andfunctionally linked one or more nucleic acids to be expressed, into themicroorganism.
 15. The method according to claim 13 or 14, wherein toincrease or cause the expression rate of a gene in microorganismscompared with the wild type ch) the specific expression activity in themicroorganism of endogenous expression units according to claim 2, whichregulate the expression of the endogenous genes, is increased comparedwith the wild type, or dh) the expression of genes in the microorganismis regulated by expression units according to claim 2 or by expressionunits according to claim 2 with increased specific expression activityaccording to embodiment a), where the genes are heterologous in relationto the expression units.
 16. The method according to claim 15, whereinthe regulation of the expression of genes in the microorganism byexpression units according to claim or by expression units according toclaim 2 with increased specific expression activity according toembodiment a) is achieved by dh1) introducing one or more expressionunits according to claim 2, where appropriate with increased specificexpression activity, into the genome of the microorganism so thatexpression of one or more endogenous genes takes place under the controlof the introduced expression units, where appropriate with increasedspecific expression activity, or dh2) introducing one or more genes intothe genome of the microorganism so that expression of one or more of theintroduced genes takes place under the control of the endogenousexpression units according to claim 2, where appropriate with increasedspecific expression activity, or dh3) introducing one or more nucleicacid constructs comprising an expression unit according to claim 2,where appropriate with increased specific expression activity, andfunctionally linked one or more nucleic acids to be expressed, into themicroorganism.
 17. The method according to claim 13 or 14, wherein toreduce the expression rate of genes in microorganisms compared with thewild type cr) the specific expression activity in the microorganism ofendogenous expression units according to claim 2, which regulate theexpression of the endogenous genes, is reduced compared with the wildtype, or dr) expression units with reduced specific expression activityaccording to embodiment cr) are introduced into the genome of themicroorganism so that expression of endogenous genes takes place underthe control of the introduced expression units with reduced expressionactivity.
 18. The method according to claim 8, wherein the genes areselected from the group of nucleic acids encoding a protein from thebiosynthetic pathway of proteinogenic and non-proteinogenic amino acids,nucleic acids encoding a protein from the biosynthetic pathway ofnucleotides and nucleosides, nucleic acids encoding a protein from thebiosynthetic pathway of organic acids, nucleic acids encoding a proteinfrom the biosynthetic pathway of lipids and fatty acids, nucleic acidsencoding a protein from the biosynthetic pathway of diols, nucleic acidsencoding a protein from the biosynthetic pathway of carbohydrates,nucleic acids encoding a protein from the biosynthetic pathway ofaromatic compounds, nucleic acids encoding a protein from thebiosynthetic pathway of vitamins, nucleic acids encoding a protein fromthe biosynthetic pathway of cofactors and nucleic acids encoding aprotein from the biosynthetic pathway of enzymes, where the genes mayoptionally comprise further regulatory elements.
 19. The methodaccording to claim 18, wherein the proteins from the biosyntheticpathway of amino acids are selected from the group of aspartate kinase,aspartate-semialdehyde dehydrogenase, diaminopimelate dehydrogenase,diaminopimelate decarboxylase, dihydrodipicolinate synthetase,dihydrodipicolinate reductase, glyceraldehyde-3-phosphate dehydrogenase,3-phosphoglycerate kinase, pyruvate carboxylase, triosephosphateisomerase, transcriptional regulator LuxR, transcriptional regulatorLysR1, transcriptional regulator LysR2, malate-quinone oxidoreductase,glucose-6-phosphate deydrogenase, 6-phosphogluconate dehydrogenase,transketolase, transaldolase, homoserine O-acetyltransferase,cystathionine gamma-synthase, cystathionine beta-lyase, serinehydroxymethyltransferase, O-acetylhomoserine sulfhydrylase,methylenetetrahydrofolate reductase, phosphoserine aminotransferase,phosphoserine phosphatase, serine acetyltransferase, homoserinedehydrogenase, homoserine kinase, threonine synthase, threonine exportercarrier, threonine dehydratase, pyruvate oxidase, lysine exporter,biotin ligase, cysteine synthase I, cysteine synthase II, coenzymeB12-dependent methionine synthase, coenzyme B12-independent methioninesynthase, sulfate adenylyltransferase subunit 1 and 2,phosphoadenosine-phosphosulfate reductase, ferredoxin-sulfite reductase,ferredoxin NADP reductase, 3-phosphoglycerate dehydrogenase, RXA00655regulator, RXN2910 regulator, arginyl-tRNA synthetase,phosphoenolpyruvate carboxylase, threonine efflux protein, serinehydroxymethyltransferase, fructose-1,6-bisphosphatase, protein ofsulfate reduction RXA077, protein of sulfate reduction RXA248, proteinof sulfate reduction RXA247, protein OpcA, 1-phosphofructokinase and6-phosphofructokinase.
 20. An expression cassette comprising a) at leastone expression unit according to claim 6 and b) at least one furthernucleic acid to be expressed, and c) where appropriate further geneticcontrol elements, where at least one expression unit and a furthernucleic acid sequence to be expressed are functionally linked together,and the further nucleic acid sequence to be expressed is heterologous inrelation to the expression unit.
 21. The expression cassette accordingto claim 20, wherein the further nucleic acid sequence to be expressedis selected from the group of nucleic acids encoding a protein from thebiosynthetic pathway of proteinogenic and non-proteinogenic amino acids,nucleic acids encoding a protein from the biosynthetic pathway ofnucleotides and nucleosides, nucleic acids encoding a protein from thebiosynthetic pathway of organic acids, nucleic acids encoding a proteinfrom the biosynthetic pathway of lipids and fatty acids, nucleic acidsencoding a protein from the biosynthetic pathway of diols, nucleic acidsencoding a protein from the biosynthetic pathway of carbohydrates,nucleic acids encoding a protein from the biosynthetic pathway ofaromatic compounds, nucleic acids encoding a protein from thebiosynthetic pathway of vitamins, nucleic acids encoding a protein fromthe biosynthetic pathway of cofactors and nucleic acids encoding aprotein from the biosynthetic pathway of enzymes.
 22. The expressioncassette according to claim 21, wherein the proteins from thebiosynthetic pathway of amino acids are selected from the group ofaspartate kinase, aspartate-semialdehyde dehydrogenase, diaminopimelatedehydrogenase, diaminopimelate decarboxylase, dihydrodipicolinatesynthetase, dihydrodipicolinate reductase, glyceraldehyde-3-phosphatedehydrogenase, 3-phosphoglycerate kinase, pyruvate carboxylase,triosephosphate isomerase, transcriptional regulator LuxR,transcriptional regulator LysR1, transcriptional regulator LysR2,malate-quinone oxidoreductase, glucose-6-phosphate deydrogenase,6-phosphogluconate dehydrogenase, transketolase, transaldolase,homoserine O-acetyltransferase, cystathionine gamma-synthase,cystathionine beta-lyase, serine hydroxymethyltransferase,O-acetylhomoserine sulfhydrylase, methylenetetrahydrofolate reductase,phosphoserine aminotransferase, phosphoserine phosphatase, serineacetyltransferase, homoserine dehydrogenase, homoserine kinase,threonine synthase, threonine exporter carrier, threonine dehydratase,pyruvate oxidase, lysine exporter, biotin ligase, cysteine synthase I,cysteine synthase II, coenzyme B12-dependent methionine synthase,coenzyme B112-independent methionine synthase activity, sulfateadenylyltransferase subunit 1 and 2, phosphoadenosine-phosphosulfatereductase, ferredoxin-sulfite reductase, ferredoxin NADP reductase,3-phosphoglycerate dehydrogenase, RXA00655 regulator, RXN2910 regulator,arginyl-tRNA synthetase, phosphoenolpyruvate carboxylase, threonineefflux protein, serine hydroxymethyltransferase,fructose-1,6-bisphosphatase, protein of sulfate reduction RXA077,protein of sulfate reduction RXA248, protein of sulfate reductionRXA247, protein OpcA, 1-phosphofructokinase and 6-phosphofructokinase.23. An expression vector comprising an expression cassette according toclaim
 20. 24. A genetically modified microorganism, where the geneticmodification leads to an alteration or causing of the transcription rateof at least one gene compared with the wild type, and is dependent on a)altering the specific promoter activity in the microorganism of at leastone endogenous nucleic acid having promoter activity according to claim1, which regulates the transcription of at least one endogenous gene, orb) regulating the transcription of genes in the microorganism by nucleicacids having promoter activity according to claim 1 or by nucleic acidshaving promoter activity according to claim 1 with altered specificpromoter activity according to embodiment a), where the genes areheterologous in relation to the nucleic acids having promoter activity.25. The genetically modified microorganism according to claim 24,wherein the regulation of the transcription of genes in themicroorganism by nucleic acids having promoter activity according toclaim 1 or by nucleic acids having promoter activity according to claim1 with altered specific promoter activity according to embodiment a), isachieved by b1) introducing one or more nucleic acids having promoteractivity according to claim 1, where appropriate with altered specificpromoter activity, into the genome of the microorganism so thattranscription of one or more endogenous genes takes place under thecontrol of the introduced nucleic acid having promoter activityaccording to claim 1, where appropriate with altered specific promoteractivity, or b2) introducing one or more genes into the genome of themicroorganism so that transcription of one or more of the introducedgenes takes place under the control of the endogenous nucleic acidshaving promoter activity according to claim 1, where appropriate withaltered specific promoter activity, or b3) introducing one or morenucleic acid constructs comprising a nucleic acid having promoteractivity according to claim 1, where appropriate with altered specificpromoter activity, and functionally linked one or more nucleic acids tobe transcribed, into the microorganism.
 26. The genetically modifiedmicroorganism according to claim 24 or 25 having increased or causedtranscription rate of at least one gene compared with the wild type,wherein ah) the specific promoter activity in the microorganism ofendogenous nucleic acids having promoter activity according to claim 1,which regulate the transcription of endogenous genes, is increasedcompared with the wild type, or bh) the transcription of genes in themicroorganism is regulated by nucleic acids having promoter activityaccording to claim 1 or by nucleic acids having increased specificpromoter activity according to embodiment ah), where the genes areheterologous in relation to the nucleic acids having promoter activity.27. The genetically modified microorganism according to claim 26,wherein the regulation of the transcription of genes in themicroorganism by nucleic acids having promoter activity according toclaim 1 or by nucleic acids having promoter activity according to claim1 with increased specific promoter activity according to embodiment a),is achieved by bh1) introducing one or more nucleic acids havingpromoter activity according to claim 1, where appropriate with increasedspecific promoter activity, into the genome of the microorganism so thattranscription of one or more endogenous genes takes place under thecontrol of the introduced nucleic acid having promoter activity, whereappropriate with increased specific promoter activity, or bh2)introducing one or more genes into the genome of the microorganism sothat transcription of one or more of the introduced genes takes placeunder the control of the endogenous nucleic acids having promoteractivity according to claim 1, where appropriate with increased specificpromoter activity, or bh3) introducing one or more nucleic acidconstructs comprising a nucleic acid having promoter activity accordingto claim 1, where appropriate with increased specific promoter activity,and functionally linked one or more nucleic acids to be transcribed,into the microorganism.
 28. The genetically modified microorganismaccording to claim 24 or 25 having reduced transcription rate of atleast one gene compared with the wild type, wherein ar) the specificpromoter activity in the microorganism of at least one endogenousnucleic acid having promoter activity according to claim 1, whichregulates the transcription of at least one endogenous gene, is reducedcompared with the wild type, or br) one or more nucleic acids havingreduced promoter activity according to embodiment a) are introduced intothe genome of the microorganism so that the transcription of at leastone endogenous gene takes place under the control of the introducednucleic acid having reduced promoter activity.
 29. A geneticallymodified microorganism, where the genetic modification leads to analteration or causing of the expression rate of at least one genecompared with the wild type, and is dependent on c) altering thespecific expression activity in the microorganism of at least oneendogenous expression unit according to claim 2, which regulates theexpression of at least one endogenous gene, compared with the wild typeor d) regulating the expression of genes in the microorganism byexpression units according to claim 2 or by expression units accordingto claim 2 with altered specific expression activity according toembodiment a), where the genes are heterologous in relation to theexpression units.
 30. The genetically modified microorganism accordingto claim 29, wherein the regulation of the expression of genes in themicroorganism by expression units according to claim 2 or by expressionunits according to claim 2 with altered specific expression activityaccording to embodiment a) is achieved by d1) introducing one or moreexpression units according to claim 2, where appropriate with alteredspecific expression activity, into the genome of the microorganism sothat expression of one or more endogenous genes takes place under thecontrol of the introduced expression units according to claim 2, whereappropriate with altered specific expression activity, or d2)introducing one or more genes into the genome of the microorganism sothat expression of one or more of the introduced genes takes place underthe control of the endogenous expression units according to claim 2,where appropriate with altered specific expression activity, or d3)introducing one or more nucleic acid constructs comprising an expressionunit according to claim 2, where appropriate with altered specificexpression activity, and functionally linked one or more nucleic acidsto be expressed, into the microorganism.
 31. The genetically modifiedmicroorganism according to claim 29 or 30 with increased or causedexpression rate of at least one gene compared with the wild type,wherein ch) the specific expression activity in the microorganism of atleast one endogenous expression unit according to claim 2, whichregulates the expression of the endogenous genes, is increased comparedwith the wild type, or dh) the expression of genes in the microorganismis regulated by expression units according to claim 2 or by expressionunits according to claim 2 with increased specific expression activityaccording to embodiment a), where the genes are heterologous in relationto the expression units.
 32. The genetically modified microorganismaccording to claim 31, wherein the regulation of the expression of genesin the microorganism by expression units according to claim 2 or byexpression units according to claim 2 with increased specific expressionactivity according to embodiment a) is achieved by dh1) introducing oneor more expression units according to claim 2, where appropriate withincreased specific expression activity, into the genome of themicroorganism so that expression of one or more endogenous genes takesplace under the control of the introduced expression unit according toclaim 2, where appropriate with increased specific expression activity,or dh2) introducing one or more genes into the genome of themicroorganism so that expression of one or more of the introduced genestakes place under the control of the endogenous expression unitsaccording to claim 2, where appropriate with increased specificexpression activity, or dh3) introducing one or more nucleic acidconstructs comprising an expression unit according to claim 2, whereappropriate with increased specific expression activity, andfunctionally linked one or more nucleic acids to be expressed, into themicroorganism.
 33. The genetically modified microorganism according toclaim 29 or 30 with reduced expression rate of at least one genecompared with the wild type, wherein cr) the specific expressionactivity in the microorganism of at least one endogenous expression unitaccording to claim 2, which regulates the expression of at least oneendogenous gene, is reduced compared with the wild type, or dr) one ormore expression units according to claim 2 with reduced expressionactivity are introduced into the genome of the microorganism so thatexpression of at least one gene takes place under the control of theintroduced expression unit according to claim 2 with reduced expressionactivity.
 34. A genetically modified microorganism comprising anexpression unit according to claim 6 and functionally linked a gene tobe expressed, where the gene is heterologous in relation to theexpression unit.
 35. The genetically modified microorganism according toclaim 34, comprising an expression cassette according to claim
 20. 36.The genetically modified microorganism according to any of claims 24 to35, wherein the genes are selected from the group of nucleic acidsencoding a protein from the biosynthetic pathway of proteinogenic andnon-proteinogenic amino acids, nucleic acids encoding a protein from thebiosynthetic pathway of nucleotides and nucleosides, nucleic acidencoding a protein from the biosynthetic pathway of organic acids,nucleic acids encoding a protein from the biosynthetic pathway of lipidsand fatty acids, nucleic acids encoding a protein from the biosyntheticpathway of diols, nucleic acids encoding a protein from the biosyntheticpathway of carbohydrates, nucleic acids encoding a protein from thebiosynthetic pathway of aromatic compounds, nucleic acids encoding aprotein from the biosynthetic pathway of vitamins, nucleic acidsencoding a protein from the biosynthetic pathway of cofactors andnucleic acids encoding a protein from the biosynthetic pathway ofenzymes, where the genes may optionally comprise further regulatoryelements.
 37. The genetically modified microorganism according to claim36, wherein the proteins from the biosynthetic pathway of amino acidsare selected from the group of aspartate kinase, aspartate-semialdehydedehydrogenase, diaminopimelate dehydrogenase, diaminopimelatedecarboxylase, dihydrodipicolinate synthetase, dihydrodipicolinatereductase, glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglyceratekinase, pyruvate carboxylase, triosephosphate isomerase, transcriptionalregulator LuxR, transcriptional regulator LysR1, transcriptionalregulator LysR2, malate-quinone oxidoreductase, glucose-6-phosphatedeydrogenase, 6-phosphogluconate dehydrogenase, transketolase,transaldolase, homoserine O-acetyltransferase, cystathioninegamma-synthase, cystathionine beta-lyase, serinehydroxymethyltransferase, O-acetylhomoserine sulfhydrylase,methylenetetrahydrofolate reductase, phosphoserine aminotransferase,phosphoserine phosphatase, serine acetyltransferase, homoserinedehydrogenase, homoserine kinase, threonine synthase, threonine exportercarrier, threonine dehydratase, pyruvate oxidase, lysine exporter,biotin ligase, cysteine synthase I, cysteine synthase II, coenzymeB132-dependent methionine synthase, coenzyme B12-independent methioninesynthase, sulfate adenylyltransferase subunit 1 and 2,phosphoadenosine-phosphosulfate reductase, ferredoxin-sulfite reductase,ferredoxin NADP reductase, 3-phosphoglycerate dehydrogenase, RXA00655regulator, RXN2910 regulator, arginyl-tRNA synthetase,phosphoenolpyruvate carboxylase, threonine efflux protein, serinehydroxymethyltransferase, fructose-1,6-bisphosphatase, protein ofsulfate reduction RXA077, protein of sulfate reduction RXA248, proteinof sulfate reduction RXA247, protein OpcA, 1-phosphofructokinase and6-phosphofructokinase.
 38. A method for preparing biosynthetic productsby cultivating genetically modified microorganisms according to any ofclaims 24 to
 37. 39. A method for preparing lysine by cultivatinggenetically modified microorganisms according to any of claims 24, 25,31 or 32, wherein the genes are selected from the group of nucleic acidsencoding an aspartate kinase, nucleic acids encoding anaspartate-semialdehyde dehydrogenase, nucleic acids encoding adiaminopimelate dehydrogenase, nucleic acids encoding a diaminopimelatedecarboxylase, nucleic acids encoding a dihydrodipicolinate synthetase,nucleic acids encoding a dihydrodipicolinate reductase, nucleic acidsencoding a glyceraldehyde-3-phosphate dehydrogenase, nucleic acidsencoding a 3-phosphoglycerate kinase, nucleic acids encoding a pyruvatecarboxylase, nucleic acids encoding a triosephosphate isomerase, nucleicacids encoding a transcriptional regulator LuxR, nucleic acids encodinga transcriptional regulator LysR1, nucleic acids encoding atranscriptional regulator LysR2, nucleic acids encoding a malate-quinoneoxidoreductase, nucleic acids encoding a glucose-6-phosphatedehydrogenase, nucleic acids encoding a 6-phosphogluconatedehydrogenase, nucleic acids encoding a transketolase, nucleic acidsencoding a transaldolase, nucleic acids encoding a lysine exporter,nucleic acids encoding a biotin ligase, nucleic acids encoding anarginyl-tRNA synthetase, nucleic acids encoding a phosphoenolpyruvatecarboxylase, nucleic acids encoding a fructose-1,6-bisphosphatase,nucleic acids encoding a protein OpcA, nucleic acids encoding a1-phosphofructokinase and nucleic acids encoding a6-phosphofructokinase.
 40. The method according to claim 39, wherein thegenetically modified microorganisms have, compared with the wild type,additionally an increased activity, of at least one of the activitiesselected from the group of aspartate kinase activity,aspartate-semialdehyde dehydrogenase activity, diaminopimelatedehydrogenase activity, diaminopimelate decarboxylase activity,dihydrodipicolinate synthetase activity, dihydrodipicolinate reductaseactivity, glyceraldehyde-3-phosphate dehydrogenase activity,3-phosphoglycerate kinase activity, pyruvate carboxylase activity,triosephosphate isomerase activity, activity of the transcriptionalregulator LuxR, activity of the transcriptional regulator LysR1,activity of the transcriptional regulator LysR2, malate-quinoneoxidoreductase activity, glucose-6-phosphate deydrogenase activity,6-phosphogluconate dehydrogenase activity, transketolase activity,transaldolase activity, lysine exporter activity, arginyl-tRNAsynthetase activity, phosphoenolpyruvate carboxylase activity,fructose-1,6-bisphosphatase activity, protein OpcA activity,1-phosphofructokinase activity, 6-phosphofructokinase activity andbiotin ligase activity.
 41. The method according to claim 39 or 40,wherein the genetically modified microorganisms have, compared with thewild type, additionally a reduced activity, of at least one of theactivities selected from the group of threonine dehydratase activity,homoserine O-acetyltransferase activity, O-acetylhomoserinesulfhydrylase activity, phosphoenolpyruvate carboxykinase activity,pyruvate oxidase activity, homoserine kinase activity, homoserinedehydrogenase activity, threonine exporter activity, threonine effluxprotein activity, asparaginase activity, aspartate decarboxylaseactivity and threonine synthase activity.
 42. A method for preparingmethionine by cultivating genetically modified microorganisms accordingto any of claims 24, 25, 31 or 32, wherein the genes are selected fromthe group of nucleic acids encoding an aspartate kinase, nucleic acidsencoding an aspartate-semialdehyde dehydrogenase, nucleic acids encodinga homoserine dehydrogenase, nucleic acids encoding aglyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a3-phosphoglycerate kinase, nucleic acids encoding a pyruvatecarboxylase, nucleic acids encoding a triosephosphate isomerase, nucleicacids encoding a homoserine O-acetyltransferase, nucleic acids encodinga cystathionine gamma-synthase, nucleic acids encoding a cystathioninebeta-lyase, nucleic acids encoding a serine hydroxymethyltransferase,nucleic acids encoding an O-acetylhomoserine sulfhydrylase, nucleicacids encoding a methylenetetrahydrofolate reductase, nucleic acidsencoding a phosphoserine aminotransferase, nucleic acids encoding aphosphoserine phosphatase, nucleic acids encoding a serineacetyltransferase, nucleic acids encoding a cysteine synthase I, nucleicacids encoding a cysteine synthase II, nucleic acids encoding a coenzymeB12-dependent methionine synthase, nucleic acids encoding a coenzymeB12-independent methionine synthase, nucleic acids encoding a sulfateadenylyltransferase, nucleic acids encoding a phosphoadenosinephosphosulfate reductase, nucleic acids encoding a ferredoxin-sulfitereductase, nucleic acids encoding a ferredoxin NADPH-reductase, nucleicacids encoding a ferredoxin activity, nucleic acids encoding a proteinof sulfate reduction RXA077, nucleic acids encoding a protein of sulfatereduction RXA248, nucleic acids encoding a protein of sulfate reductionRXA247, nucleic acids encoding an RXA0655 regulator and nucleic acidsencoding an RXN2910 regulator.
 43. The method according to claim 42,wherein the genetically modified microorganisms have, compared with thewild type, additionally an increased activity, of at least one of theactivities selected from the group of aspartate kinase activity,aspartate-semialdehyde dehydrogenase activity, homoserine dehydrogenaseactivity, glyceraldehyde-3-phosphate dehydrogenase activity,3-phosphoglycerate kinase activity, pyruvate carboxylase activity,triosephosphate isomerase activity, homoserine O-acetyltransferaseactivity, cystathionine gamma-synthase activity, cystathioninebeta-lyase activity, serine hydroxymethyltransferase activity,O-acetylhomoserine sulfhydrylase activity, methylenetetrahydrofolatereductase activity, phosphoserine aminotransferase activity,phosphoserine phosphatase activity, serine acetyltransferase activity,cysteine synthase I activity, cysteine synthase II activity, coenzymeB12-dependent methionine synthase activity, coenzyme B112-independentmethionine synthase activity, sulfate adenylyltransferase activity,phosphoadenosine-phosphosulfate reductase activity, ferredoxin-sulfitereductase activity, ferredoxin NADPH-reductase activity, ferredoxinactivity, activity of a protein of sulfate reduction RXA077, activity ofa protein of sulfate reduction RXA248, activity of a protein of sulfatereduction RXA247, activity of an RXA655 regulator and activity of anRXN2910 regulator.
 44. The method according to claim 42 or 43, whereinthe genetically modified microorganisms have, compared with the wildtype, additionally a reduced activity, of at least one of the activitiesselected from the group of homoserine kinase activity, threoninedehydratase activity, threonine synthase activity, meso-diaminopimelateD-dehydrogenase activity, phosphoenolpyruvate carboxykinase activity,pyruvate oxidase activity, dihydrodipicolinate synthase activity,dihydrodipicolinate reductase activity and diaminopicolinatedecarboxylase activity.
 45. A method for preparing threonine bycultivating genetically modified microorganisms according to any ofclaims 24, 25, 31 or 32, wherein the genes are selected from the groupof nucleic acids encoding an aspartate kinase, nucleic acids encoding anaspartate-semialdehyde dehydrogenase, nucleic acids encoding aglyceraldehyde-3-phosphate dehydrogenase, nucleic acids encoding a3-phosphoglycerate kinase, nucleic acids encoding a pyruvatecarboxylase, nucleic acids encoding a triosephosphate isomerase, nucleicacids encoding a homoserine kinase, nucleic acids encoding a threoninesynthase, nucleic acids encoding a threonine exporter carrier, nucleicacids encoding a glucose-6-phosphate dehydrogenase, nucleic acidsencoding a transaldolase, nucleic acids encoding a transketolase,nucleic acids encoding a malate-quinone oxidoreductase, nucleic acidsencoding a 6-phosphogluconate dehydrogenase, nucleic acids encoding alysine exporter, nucleic acids encoding a biotin ligase, nucleic acidsencoding a phosphoenolpyruvate carboxylase, nucleic acids encoding athreonine efflux protein, nucleic acids encoding afructose-1,6-bisphosphatase, nucleic acids encoding an OpcA protein,nucleic acids encoding a 1-phosphofructokinase, nucleic acids encoding a6-phosphofructokinase, and nucleic acids encoding a homoserinedehydrogenase.
 46. The method according to claim 45, wherein thegenetically modified microorganisms have, compared with the wild type,additionally an increased activity, of at least one of the activitiesselected from the group of aspartate kinase activity,aspartate-semialdehyde dehydrogenase activity,glyceraldehyde-3-phosphate dehydrogenase activity, 3-phosphoglyceratekinase activity, pyruvate carboxylase activity, triosephosphateisomerase activity, threonine synthase activity, activity of a threonineexport carrier, transaldolase activity, transketolase activity,glucose-6-phosphate dehydrogenase activity, malate-quinoneoxidoreductase activity, homoserine kinase activity, biotin ligaseactivity, phosphoenolpyruvate carboxylase activity, threonine effluxprotein activity, protein OpcA activity, 1-phosphofructokinase activity,6-phosphofructokinase activity, fructose-1-6-bisphosphatase activity,6-phosphogluconate dehydrogenase and homoserine dehydrogenase activity.47. The method according to claim 45 or 46, wherein the geneticallymodified microorganisms have, compared with the wild type, additionallya reduced activity, of at least one of the activities selected from thegroup of threonine dehydratase activity, homoserine O-acetyltransferaseactivity, serine hydroxymethyltransferase activity, O-acetylhomoserinesulfhydrylase activity, meso-diaminopimelate D-dehydrogenase activity,phosphoenolpyruvate carboxykinase activity, pyruvate oxidase activity,dihydrodipicolinate synthetase activity, dihydrodipicolinate reductaseactivity, asparaginase activity, aspartate decarboxylase activity,lysine exporter activity, acetolactate synthase activity, ketol-acidreductoisomerase activity, branched chain aminotransferase activity,coenzyme B12-dependent methionine synthase activity, coenzymeB12-independent methionine synthase activity, dihydroxy-acid dehydrataseactivity and diaminopicolinate decarboxylase activity.
 48. The methodaccording to any of claims 38 to 47, wherein the biosynthetic productsare isolated and, where appropriate, purified from the cultivationmedium after and/or during the cultivation step.
 49. (canceled) 50.(canceled)
 51. An expression unit which enables genes to be expressed inbacteria of the genus Corynebacterium or Brevibacterium, comprising atleast one of the nucleic acid sequences of SEQ ID NOs:42, 43 or
 44. 52.The expression unit according to claim 51, wherein the nucleic acidsequence of SEQ ID NO:44 is used as a ribosome binding site. 53.(canceled)
 54. The expression unit according to claim 51, wherein one ofthe nucleic acid sequences of SEQ ID NOs: 42 or 43 is used as a −10region.